Loss of cold tolerance is conferred by absence of the WRKY34 promoter fragment during tomato evolution
番茄进化过程中 WRKY34 启动子片段的缺失会导致耐寒性丧失

The divergence of gene function, primarily driven by mutation, gene duplication, and gene loss, is fundamental to evolutionary processes^1,2. Such divergence in gene function may be caused by mutations in coding regions that alter protein function. For instance, a 45 bp deletion in the ZmRR1 coding region prevents its phosphorylation by ZmMPK8, inhibiting its degradation via the 26S proteasome pathway and thereby enhancing maize cold tolerance³. Alternatively, divergence in gene function may also result from mutations in cis-regulatory regions, which interact intricately to shape expression patterns across different tissues during development⁴. For example, the CRISPR/Cas9 cis-regulatory allelic series of tomato SlWOX9 reveals that different pleiotropic functions can be mapped to specific cis-regulatory regions⁵. Evolutionary innovations in transcriptional regulation often result from changes in cis-regulatory regions, where abundant sequences directly binding to transcription factors offer significant mutational potential to alter gene expression and phenotypes⁶. For instance, a crucial variation in the W-box motif within the SlWRKY33 promoter suppresses its self-transcriptional activity in response to cold stress, thus contributing to the cold sensitivity observed in cultivated tomatoes compared to cold-tolerant wild tomatoes⁷. Nevertheless, our comprehension of the alterations and roles of cis-regulatory regions during crop evolution, as well as their regulatory mechanisms, remains limited.
基因功能的分化主要由突变、基因复制和基因丢失驱动，是进化过程的基础^1,2。这种基因功能的分化可能是由改变蛋白质功能的编码区突变引起的。例如，ZmRR1 编码区 45 bp 的缺失会阻止其被 ZmMPK8 磷酸化，从而抑制其通过 26S 蛋白酶体途径的降解，从而增强玉米耐寒性³。或者，基因功能的分化也可能是由顺式调控区的突变引起的，这些区在发育过程中错综复杂地相互作用以形成不同组织的表达模式⁴。例如，番茄 SlWOX9 的 CRISPR/Cas9 顺式调节等位基因系列揭示了不同的多效性功能可以映射到特定的顺式调节区域⁵。转录调控的进化创新通常是由顺式调控区域的变化引起的，其中直接与转录因子结合的大量序列为改变基因表达和表型提供了显著的突变潜力⁶。例如，SlWRKY33 启动子内 W-box 基序的关键变异抑制了其响应寒冷胁迫的自我转录活性，从而导致与耐寒野生番茄相比，在栽培番茄中观察到的寒冷敏感性⁷。然而，我们对作物进化过程中顺式调控区域的改变和作用以及它们的调控机制的理解仍然有限。

In recent years, our understanding of transcriptional regulation has broadened to include the level of chromatin structure⁸. Chromatin remodeling, which involves the dynamic modification of chromatin structure, plays a crucial role in controlling the accessibility of transcriptional machinery to DNA⁹. In the medical field, chromatin remodeling has been extensively studied for its impact on cell differentiation, organ development, and its involvement in diseases such as cancer^10,11. For instance, the chromatin remodeler CHD6 promotes colorectal cancer development by regulating TMEM65-mediated mitochondrial dynamics¹². In botany, chromatin remodeling is increasingly recognized as pivotal in how plants respond to environmental cues. Several studies have indicated that environmental stress can alter chromatin structure, thereby influencing transcriptional regulation^13,14. For example, heat stress triggers genome-wide chromatin accessibility changes in tomato, with HSFA1 binding promoting the formation of promoter-enhancer contacts to drive the expression of heat stress-responsive genes¹⁵. Similarly, cold stress enhances chromatin accessibility and leads to bivalent histone modifications of active genes in potato¹⁶. Moreover, a lamin-like protein OsNMCP1 in rice modifies chromatin accessibility by interacting with a chromatin remodeler OsSWI3C, thereby regulating numerous genes involved in root growth and drought response¹⁷. Hence, changes in chromatin structure may serve as the initial step in initiating transcriptional stress responses. Nevertheless, the mechanisms and extent to which environmental stress induces chromatin dynamics remain largely unknown. Additionally, the causal relationship between chromatin dynamics and transcriptional responses under environmental stress requires further elucidation.
近年来，我们对转录调控的理解已经扩大到包括染色质结构水平⁸。染色质重塑涉及染色质结构的动态修饰，在控制转录机制对 DNA 的可及性方面起着至关重要的作用⁹。在医学领域，染色质重塑因其对细胞分化、器官发育的影响以及与癌症等疾病的关系而得到广泛研究^10,11。例如，染色质重塑基因 CHD6 通过调节 TMEM65 介导的线粒体动力学促进结直肠癌的发展¹²。在植物学中，染色质重塑越来越被认为是植物如何响应环境线索的关键。几项研究表明，环境应激会改变染色质结构，从而影响转录调控^13,14。例如，热应激会触发番茄的全基因组染色质可及性变化，HSFA1 结合会促进启动子-增强子接触的形成，从而驱动热应激反应基因的表达¹⁵。同样，冷应激增强了染色质的可及性，并导致马铃薯活性基因的二价组蛋白修饰¹⁶。此外，水稻中的核纤层蛋白样蛋白 OsNMCP1 通过与染色质重塑基因 OsSWI3C 相互作用来修饰染色质的可及性，从而调节参与根系生长和干旱反应的众多基因¹⁷。因此，染色质结构的变化可以作为启动转录应激反应的初始步骤。 然而，环境应激诱导染色质动力学的机制和程度在很大程度上仍然未知。此外，在环境压力下，染色质动力学和转录反应之间的因果关系需要进一步阐明。

The SWIB/MDM2 domain superfamily of proteins comprises a group of proteins characterized by the presence of the SWIB (SWI/SNF complex BCL7/BCL7A interacting domain) and/or MDM2 (Mouse Double Minute 2 homolog) domains^18,19. These proteins are evolutionarily conserved and exist in various eukaryotes^20,21. Numerous studies have highlighted the critical role of SWIB/MDM2 domain proteins in diverse cellular processes, particularly in chromatin remodeling and gene expression regulation²⁰. For example, the SWP73 protein, a member of the SWIB/MDM2 domain superfamily, is essential for supporting yeast growth at elevated temperatures. Additionally, it plays a pivotal role in repressing seedling growth by modulating chromatin accessibility of genes regulating hypocotyl cell size in Arabidopsis^22,23. Furthermore, a study underscores the importance of SWIB-4, a SWIB domain protein in spinach chloroplasts, which not only structures the nucleoid core but also binds DNA via its histone H1 motif, thus playing a crucial role in the compaction and regulation of chloroplast DNA²⁴. Nevertheless, the functions of these proteins in plants, especially their ability for direct DNA binding in the nucleus, remain largely unexplored.
SWIB/MDM2 结构域蛋白质超家族由一组蛋白质组成，其特征是存在 SWIB（SWI/SNF 复合物 BCL7/BCL7A 相互作用结构域）和/或 MDM2（小鼠双分钟 2 同源物）结构域^18,19。这些蛋白质在进化上是保守的，存在于各种真核生物中^20,21。大量研究强调了 SWIB/MDM2 结构域蛋白在不同细胞过程中的关键作用，特别是在染色质重塑和基因表达调控中²⁰。例如，SWP73 蛋白是 SWIB/MDM2 结构域超家族的成员，对于支持酵母在高温下的生长至关重要。此外，它通过调节拟南芥下胚轴细胞大小的基因的染色质可及性，在抑制幼苗生长中起关键作用 ^22,23。此外，一项研究强调了 SWIB-4 的重要性，SWIB-4 是菠菜叶绿体中的 SWIB 结构域蛋白，它不仅构建了类核核心，还通过其组蛋白 H1 基序结合 DNA，从而在叶绿体 DNA 的压缩和调节中起着至关重要的作用²⁴。然而，这些蛋白质在植物中的功能，尤其是它们在细胞核中直接结合 DNA 的能力，在很大程度上仍未得到探索。

Cold stress poses significant threats to crops, leading to reduced growth, impaired development, and lower yields. Plants have evolved various pathways to withstand cold stress, including the CBF-COR pathway and hormonal pathways²⁵. In the CBF-COR pathway, ICE (Inducer of CBF Expression) proteins act as upstream regulators, activating C-repeat binding factors (CBFs), which in turn induce the expression of cold-responsive (COR) genes to enhance cold tolerance²⁶. Different subspecies within a species often exhibit distinct cold tolerances due to evolutionary adaptations to their specific environments. For example, a study employing a combination of genetic mapping and gene expression analysis revealed that temperate japonica rice varieties have evolved lower expression of HAN1 gene due to an increase in MYB cis-elements within its promoter during domestication. This adaptation enhances chilling tolerance mediated by jasmonic acid (JA), aiding in the adaptation to a temperate climate²⁷. Similarly, using a metabolite genome-wide association study (mGWAS), variations in the ZmICE1 promoter were identified to affect its interaction with ZmMYB39, thereby influencing cold tolerance in maize through the regulation of metabolic reprogramming and COR gene expression²⁸. Therefore, it is of great importance to utilize multi-omics analysis to identify key genetic loci for cold tolerance in crops. Different wild tomatoes have different levels of cold tolerance, and Solanum habrochaites is considered to be one of the most cold tolerant wild tomatoes²⁹.
寒冷胁迫对作物构成重大威胁，导致生长缓慢、发育受损和产量降低。植物已经进化出多种途径来承受寒冷胁迫，包括 CBF-COR 途径和激素途径²⁵。在 CBF-COR 通路中，ICE（CBF 表达诱导剂）蛋白充当上游调节因子，激活 C 重复序列结合因子 （CBF），进而诱导冷反应 （COR） 基因的表达以增强耐寒性²⁶。由于进化适应其特定环境，物种内的不同亚种通常表现出不同的耐寒性。例如，一项采用遗传图谱和基因表达分析相结合的研究显示，由于在驯化过程中其启动子内的 MYB 顺式元件增加，温带粳稻品种的 HAN1 基因表达较低。这种适应增强了茉莉酸 （JA） 介导的耐寒性，有助于适应温带气候²⁷。同样，使用代谢物全基因组关联研究 （mGWAS），确定 ZmICE1 启动子的变异会影响其与 ZmMYB39 的相互作用，从而通过调节代谢重编程和 COR 基因表达来影响玉米的耐寒性²⁸。因此，利用多组学分析来确定作物耐寒的关键遗传位点非常重要。不同的野生西红柿具有不同的耐寒程度，而 Solanum habrochaites 被认为是最耐寒的野生西红柿之一²⁹。

In this study, through the combined analysis of transposase-accessible chromatin sequencing (ATAC-Seq) and transcriptome sequencing (RNA-Seq), we observe that expression of WRKY34 remains largely unchanged following cold treatment in cold-sensitive cultivated tomato S. lycopersicum. Conversely, in cold-tolerant wild tomato S. habrochaites, exposure to cold leads to transcriptional suppression of WRKY34, accompanied by chromatin opening. Importantly, we identify a 60 bp InDel in the WRKY34 promoter that affects its binding to SWIBs and transcription factor GATA29, thereby influencing its chromatin accessibility and expression level under cold stress. Additionally, we demonstrate that SWIBs and GATA29 interact with each other to cooperatively suppress the expression of WRKY34. Furthermore, WRKY34 interferes with the CBF-COR cold response pathway through interaction with CBF1 or direct transcriptional inhibition, thereby negatively regulating cold tolerance. Our study elucidates that polymorphisms in cis-regulatory regions leading to differences in chromatin structure and gene expression during crop evolution, providing insights into the natural regulatory mechanism of cold tolerance in tomato.
在这项研究中，通过转座酶可及染色质测序 （ATAC-Seq） 和转录组测序 （RNA-Seq） 的联合分析，我们观察到 WRKY34 的表达在冷处理后在冷处理后基本保持不变 S. lycopersicum.相反，在耐寒的野生番茄 S. habrochaites 中，暴露于寒冷会导致 WRKY34 的转录抑制，并伴有染色质开放。重要的是，我们在 WRKY34 启动子中鉴定了一个 60 bp 的 InDel，它影响其与 SWIB 和转录因子 GATA29 的结合，从而影响其在低温胁迫下的染色质可及性和表达水平。此外，我们证明 SWIBs 和 GATA29 相互作用以协同抑制 WRKY34 的表达。此外，WRKY34 通过与 CBF1 相互作用或直接转录抑制来干扰 CBF-COR 冷反应通路，从而负向调节耐冷性。我们的研究阐明了顺式调控区的多态性导致作物进化过程中染色质结构和基因表达的差异，为番茄耐寒的自然调控机制提供了见解。

A potential role of WRKY34 in tomato response to cold stress
WRKY34 在番茄对寒冷胁迫的反应中的潜在作用

Previous research has shown substantial difference in cold tolerance between cultivated tomato and various wild tomato species⁷. To delve deeper into the distinctions in the potential regulatory mechanisms of cold tolerance between wild and cultivated tomatoes, we exposed the cold-sensitive cultivated tomato (Solanum lycopersicum) Ailsa Craig (AC) and the cold-tolerant wild tomato (S. habrochaites) LA1777 to cold stress for 6 h, followed by ATAC-Seq and RNA-Seq analyses, respectively. ATAC-Seq data analysis revealed that genome-wide chromatin accessibility decreased rather than increased in both wild and cultivated tomatoes after cold treatment (Fig. 1a and Supplementary Fig. 1a). High Spearman correlation coefficients between biological replicates indicate the reliability of ATAC-Seq results (Supplementary Fig. 1b). Genes near peaks exhibiting decreased chromatin accessibility (closing chromatin regions) under cold stress in both cultivated tomato AC and wild tomato LA1777 were predominantly enriched in pathways related to growth and development, such as photosynthesis, starch and sucrose metabolism and amino acid metabolism (Supplementary Fig. 1c). However, under cold stress conditions, the enrichment ratio of chromatin accessibility peaks in wild tomato LA1777 was significantly higher than that in cultivated tomato AC (Supplementary Fig. 1d). Furthermore, genes associated with differential chromatin accessibility peaks (DCAPs) between the two accessions were primarily enriched in gene ontology (GO) terms such as DNA binding, organic cyclic compound binding and heterocyclic compound binding (Supplementary Fig. 1e). Analysis of ATAC-Seq data revealed 7082 genes associated with DCAPs between the two accessions (AC and LA1777) under cold stress conditions (Fig. 1b and Supplementary Data 1), along with 337 genes showing increased chromatin accessibility peaks (opening chromatin regions) in wild tomato LA1777 post cold treatment (Fig. 1b and Supplementary Data 2). Analysis of RNA-Seq data revealed 3434 differentially expressed genes (DEGs) in wild tomato LA1777 under cold stress, whereas 25557 genes in cultivated tomato AC exhibited no significant difference under cold stress (Fig. 1b and Supplementary Data 3, 4).
先前的研究表明，培育番茄和各种野生番茄品种在耐寒性方面存在很大差异⁷。为了更深入地研究野生和栽培番茄之间耐寒性的潜在调控机制的区别，我们将对寒冷敏感的栽培番茄 （Solanum lycopersicum） Ailsa Craig （AC） 和耐寒的野生番茄 （S. habrochaites） LA1777 暴露于冷胁迫 6 小时，然后分别进行 ATAC-Seq 和 RNA-Seq 分析。ATAC-Seq 数据分析显示，冷处理后，野生和栽培西红柿的全基因组染色质可及性降低而不是增加（图 D）。1a 和补充图生物学重复之间的高 Spearman 相关系数表明 ATAC-Seq 结果的可靠性（补充图 D）。1b）. 在培育番茄 AC 和野生番茄 LA1777 中，在低温胁迫下表现出染色质可及性降低（关闭染色质区域）的峰值附近的基因主要富集在与生长发育相关的途径中，例如光合作用、淀粉和蔗糖代谢以及氨基酸代谢（补充图 D）。然而，在低温胁迫条件下，野生番茄 LA1777 中染色质可及性峰的富集率显著高于栽培番茄 AC（补充图 D）。此外，与两个种质之间的差异染色质可及性峰 （DCAP） 相关的基因主要富集在基因本体论 （GO） 术语中，例如 DNA 结合、有机环化合物结合和杂环化合物结合（补充图 D）。1e）。 ATAC-Seq 数据分析显示，在低温胁迫条件下，两个种质（AC 和 LA1777）之间有 7082 个基因与 DCAP 相关（图 D）。1b 和补充数据 1），以及 337 个基因显示冷处理后野生番茄 LA1777 染色质可及性峰值（打开染色质区域）增加（图 1）。1b 和补充数据 2）。RNA-Seq 数据分析显示，在低温胁迫下，野生番茄 LA1777 中有 3434 个差异表达基因 （DEG），而栽培番茄 AC 中的 25557 个基因在低温胁迫下没有表现出显著差异（图 D）。1b 和补充数据 3、4）。

a Heatmaps of ATAC-Seq read density in all samples (two replicates per sample) within a 3 kb window centered on transcriptional start sites (TSS). Detected accessible regions are showed. Each row represents one peak. The color represents the intensity of chromatin accessibility. b Venn diagram illustrating cold response candidate genes identified by combined ATAC-Seq and RNA-Seq analysis. Categories include: ATAC-Seq (7082) LA1777_4vsAC_4_Diff, indicating 7082 differential chromatin accessibility peaks (DCAPs)-associated genes between the two accessions (cultivated tomato AC and wild tomato LA1777) under cold stress conditions identified by ATAC-Seq data; ATAC-Seq (337) LA1777_4vsLA1777_25_Up, indicating 337 increased chromatin accessibility peaks (opening chromatin regions) related genes in wild tomato LA1777 after cold stress identified by ATAC-Seq; RNA-Seq (25557) AC_Insig, indicating 25557 genes with no significant change in expression in cultivated tomato AC after cold stress identified by RNA-Seq data; RNA-Seq (3434) LA1777_Sig, indicating 3434 differentially expressed genes (DEGs) in wild tomato LA1777 under cold stress identified by RNA-Seq data. c Expression levels of SlWRKY34 in S. lycopersicum (Ailsa Craig, AC/SlW34) and ShWRKY34 in S. habrochaites (LA1777/ShW34) plants under cold stress. Total RNA was isolated from leaf samples at the indicated times. d WRKY34 protein accumulation in AC and LA1777 under cold stress. The Actin protein was used as a loading control. The experiments were repeated three times with similar results (c, d). Values are expressed as means ± SD, n = 3 (*P < 0.05 and ***P < 0.001; two-tailed Student’s t-test). AC_25, cultivated tomato AC under normal temperature (25 °C); AC_4, cultivated tomato AC under cold stress (4 °C); LA1777_25, wild tomato LA1777 under normal temperature (25 °C); LA1777_4, wild tomato LA1777 under cold stress (4 °C). Source data are provided as a Source Data file.
a 以转录起始位点 （TSS） 为中心的 3 kb 窗口内所有样品（每个样品两次重复）中 ATAC-Seq 读取密度的热图。将显示检测到的可访问区域。每行代表一个峰。颜色代表染色质可及性的强度。b 维恩图说明了通过 ATAC-Seq 和 RNA-Seq 联合分析鉴定的冷反应候选基因。类别包括：ATAC-Seq （7082） LA1777_4vsAC_4_Diff，表明在 ATAC-Seq 数据鉴定的低温胁迫条件下，两个种质（培养番茄 AC 和野生番茄 LA1777）之间存在 7082 个差异染色质可及性峰 （DCAP） 相关基因;ATAC-Seq （337） LA1777_4vsLA1777_25_Up，表明 ATAC-Seq 鉴定后野生番茄 LA1777 中 337 个染色质可及性峰（打开染色质区域）相关基因增加;RNA-Seq （25557） AC_Insig，表明 RNA-Seq 数据鉴定的低温胁迫后培养番茄 AC 中有 25557 个基因的表达没有显着变化;RNA-Seq （3434） LA1777_Sig，表明 RNA-Seq 数据在低温胁迫下野生番茄 LA1777 中存在 3434 个差异表达基因 （DEGs）。c 低温胁迫下石松 （Ailsa Craig， AC/SlW34） 中 SlWRKY34 和 S. habrochaites （LA1777/ShW34） 植物中 ShWRKY34 的表达水平。在指定时间从叶片样品中分离总 RNA。d 寒冷胁迫下 WRKY34 蛋白在 AC 和 LA1777 中的积累。肌动蛋白用作上样对照。实验重复 3 次，结果相似 （c， d）。值表示为 SD ±平均值，n = 3（*P < 0.05 和 ***P < 0。001;双尾学生 t 检验）。AC_25、常温 （25 °C） 下培养的番茄 AC;AC_4， 冷胁迫 （4 °C） 下栽培的番茄 AC;LA1777_25、常温 （25 °C） 下野生番茄 LA1777;LA1777_4，冷应激 （4 °C） 下的野生番茄 LA1777。源数据作为 源数据 文件提供。

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To further identify candidate genes responsible for cold tolerance in tomato, we integrated the ATAC-Seq and RNA-Seq data as described above and generated a Venn diagram (Fig. 1b). The intersection of the Venn diagram revealed that the expression levels of 18 genes remained unchanged in the cold-sensitive cultivated tomato AC after cold treatment, but showed differential expression in the cold-tolerant wild tomato LA1777 after cold treatment. Interestingly, chromatin accessibilities of these 18 genes were significantly different between the two accessions (AC and LA1777) under cold stress, with chromatin opening observed in the cold-tolerant wild tomato LA1777 after cold stress (Fig. 1b and Supplementary Data 5). The expression of these 18 genes was validated through RT-qPCR analysis, confirming the reliability of the RNA-Seq data (Supplementary Fig. 2a). Additionally, RT-qPCR validation was performed on three genes from the RNA-Seq data that exhibited significant expression changes after cold treatment in AC but no difference in LA1777, as well as three genes that showed no difference in expression after cold treatment in both AC and LA1777 (Supplementary Fig. 2b, c). The expression patterns of these genes validated by RT-qPCR were consistent with those observed in the RNA-Seq data. Subsequently, we analyzed the 18 genes that displayed differential expression patterns and chromatin accessibilities between wild and cultivated tomatoes after cold stress. These genes, including MAPKKK86, transcription factor WRKY34, phosphatidylinositol transfer protein SFH5, and chaperone protein dnaJ, among others, play diverse roles in plant responses to cold stress, encompassing signal transduction, gene transcription, DNA repair, metabolism regulation, protein processing, and cell structure maintenance (Supplementary Data 5). The differential accessibility regions of these 18 genes were primarily located in distal intergenic regions, with four of these genes exhibiting significantly induced expression levels in LA1777 after cold treatment, while the expression of fourteen genes was significantly down-regulated following cold treatment in LA1777 (Supplementary Fig. 3 and Supplementary Data 5).
为了进一步确定负责番茄耐寒性的候选基因，我们如上所述整合了 ATAC-Seq 和 RNA-Seq 数据并生成了维恩图（图 D）。1b）. 维恩图交集显示，冷处理后，18个基因在冷处理后在冷敏栽培番茄AC中表达水平保持不变，但在冷处理后在耐寒野生番茄LA1777中表现出差异表达。有趣的是，在低温胁迫下，这两个种质（AC 和 LA1777）的染色质可及性在两个种质（AC 和 LA1777）之间显著不同，在低温胁迫后在耐寒野生番茄 LA1777 中观察到染色质开放（图 D）。1b 和补充数据 5）。通过 RT-qPCR 分析验证了这 18 个基因的表达，证实了 RNA-Seq 数据的可靠性（补充图 1）。此外，对 RNA-Seq 数据中的 3 个基因进行了 RT-qPCR 验证，这些基因在 AC 中冷处理后表达变化显著，但在 LA1777 中表达没有差异，以及 3 个基因在 AC 和 LA1777 冷处理后表达没有差异（补充图 D）。2b， c） 的通过 RT-qPCR 验证的这些基因的表达模式与 RNA-Seq 数据中观察到的一致。随后，我们分析了 18 个基因，这些基因在冷应激后表现出野生和栽培番茄之间不同的表达模式和染色质可及性。 这些基因，包括 MAPKKK86、转录因子 WRKY34、磷脂酰肌醇转移蛋白 SFH5 和伴侣蛋白 dnaJ 等，在植物对寒冷胁迫的反应中发挥着多种作用，包括信号转导、基因转录、DNA 修复、代谢调节、蛋白质加工和细胞结构维持（补充数据 5).这 18 个基因的差异可及性区域主要位于远端基因间区域，其中 4 个基因在冷处理后在 LA1777 中表现出显著诱导的表达水平，而在 LA1777 中冷处理后，14 个基因的表达显著下调（补充图 D）。3 和补充数据 5）。

Chromatin accessibility of genes, particularly in the promoter regions, can directly impact gene transcriptional activity³⁰. Previous studies have shown that many crucial regulatory elements, such as binding sites for most transcription factors, are typically situated within 3 kb of gene promoters^31,32. Consequently, we directed our attention to genes whose differential chromatin accessibility regions were located within the 3 kb promoter region. We identified only one gene, WRKY34, that not only exhibited a differential accessibility region within 3 kb of the promoter between two accessions (AC and LA1777) under cold stress, but also corresponded to the chromatin opening region in LA1777 after cold treatment within 3 kb of the promoter (Supplementary Fig. 3 and Supplementary Data 5). The genome browser view of ATAC-Seq results for WRKY34 illustrated that the chromatin surrounding WRKY34 was predominantly closed in both AC and LA1777 plants under normal conditions. However, while chromatin remained closed in AC plants after cold stress, the chromatin within 2-3 kb upstream of the WRKY34 promoter noticeably opened in LA1777 plants after cold stress (Supplementary Fig. 3). Furthermore, the transcripts of WRKY34 exhibited minimal change in cold-sensitive cultivated tomato AC, but were significantly down-regulated in cold-tolerant wild tomato LA1777 under cold stress (Fig. 1c and Supplementary Data 5). Additionally, consistent with the transcript levels of WRKY34, we observed that WRKY34 protein accumulation in cultivated tomato AC leaves remained largely unaffected by cold stress, whereas the abundance of WRKY34 protein in wild tomato LA1777 leaves gradually declined with increasing duration of cold treatment (Fig. 1d). These results suggest that transcription factor WRKY34 may play a potential role in regulating tomato cold tolerance.
基因的染色质可及性，尤其是在启动子区域，可直接影响基因转录活性³⁰。先前的研究表明，许多关键的调节元件，例如大多数转录因子的结合位点，通常位于基因启动子^31,32 的 3 kb 范围内。因此，我们将注意力集中在差异染色质可及性区域位于 3 kb 启动子区域内的基因上。我们只鉴定出一个基因 WRKY34，它不仅在冷应激下两个种质（AC 和 LA1777）之间在启动子 3 kb 范围内表现出差异可及性区域，而且在冷处理后 LA1777 中启动子 3 kb 以内的染色质开放区域也对应（补充图 D）。3 和补充数据 5）。WRKY34 的 ATAC-Seq 结果的基因组浏览器视图表明，在正常条件下，WRKY34 周围的染色质在 AC 和 LA1777 植物中都主要是闭合的。然而，虽然在冷应激后 AC 植物中染色质保持闭合，但在冷应激后，WRKY34 启动子上游 2-3 kb 内的染色质在 LA1777 植物中明显开放（补充图 D）。3）. 此外，WRKY34 的转录物在寒冷敏感的栽培番茄 AC 中表现出微小的变化，但在寒冷胁迫下，在耐寒的野生番茄 LA1777 中显着下调（图 D）。1c 和补充数据 5）。 此外，与 WRKY34 的转录水平一致，我们观察到 WRKY34 蛋白在栽培番茄 AC 叶片中的积累在很大程度上不受低温胁迫的影响，而野生番茄 LA1777 叶片中 WRKY34 蛋白的丰度随着冷处理时间的增加而逐渐下降（图 D）。这些结果表明，转录因子 WRKY34 可能在调节番茄耐寒性中发挥潜在作用。

WRKY34 negatively regulates cold tolerance of tomato
WRKY34 负调控番茄的耐寒性

Both cultivated tomato SlWRKY34 and wild tomato ShWRKY34 are situated at the end of chromosome 5 in their respective genomes. To investigate the function of WRKY34 alleles in response to cold stress in wild and cultivated tomatoes, we introduced S. habrochaites introgression line LA3942, which carries ShWRKY34 instead of SlWRKY34, along with its recurrent parent S. lycopersicum LA4024 and donor parent S. habrochaites LA1777 (Supplementary Fig. 4). We silenced SlWRKY34 (TRV-SlW34), ShWRKY34 (TRV-ShW34) in LA4024, LA3942 and LA1777 plants using virus-induced gene silencing (VIGS) technique, respectively. Silencing efficiency exceeded 65%, resulting in significantly reduced WRKY34 expression levels in silenced seedlings compared to non-silenced seedlings (TRV) (Fig. 2a). After cold stress, ShWRKY34 expression in TRV control and ShWRKY34-silenced seedlings (TRV-ShW34) of LA3942 and LA1777 was decreased significantly, while SlWRKY34 expression in TRV and SlWRKY34-silenced seedlings (TRV-SlW34) of LA4024 showed no significant difference compared to their respective control plants (Fig. 2a). Under normal conditions, no discernible phenotype change was observed in seedlings after WRKY34 silencing (Fig. 2b). Notably, TRV seedlings of LA3942 and LA1777, containing the ShWRKY34 gene, exhibited greater tolerance to cold stress than TRV seedlings of LA4024, which contains the SlWRKY34 gene. This was evidenced by lower relative electrolyte leakage (REL), higher maximum photochemical efficiency of photosystem II (Fv/Fm) and higher survival rate under cold stress (Fig. 2b–d and Supplementary Fig. 5a). Silencing of ShWRKY34 in LA3942 and LA1777 still maintained strong cold tolerance in tomato seedlings. Importantly, silencing of SlWRKY34 in LA4024 significantly increased cold tolerance, resulting in lower REL, higher Fv/Fm and higher survival rate compared with its TRV under cold stress (Fig. 2b–d and Supplementary Fig. 5a).
培养的番茄 SlWRKY34 和野生番茄 ShWRKY34 都位于各自基因组中 5 号染色体的末端。为了研究野生和栽培西红柿 WRKY34 等位基因对低温胁迫的响应功能，我们引入了 S. habrochaites 渗入系 LA3942，它携带 ShWRKY34 而不是 SlWRKY34，以及其递归亲本 S. lycopersicum LA4024 和供体亲本 S. habrochaites LA1777（补充图 D）。4）. 我们分别使用病毒诱导的基因沉默 （VIGS） 技术在 LA4024、LA3942 和 LA1777 植物中沉默了 SlWRKY34 （TRV-SlW34） 和 ShWRKY34 （TRV-ShW34）。沉默效率超过 65%，与非沉默幼苗 （TRV） 相比，沉默幼苗中 WRKY34 的表达水平显著降低（图 D）。2a）. 冷胁迫后，ShWRKY34 在 TRV 对照和 LA3942 和 LA1777 的 ShWRKY34 沉默幼苗 （TRV-ShW34） 中的表达显著降低，而 SlWRKY34 在 TRV 和 LA4024 的 SlWRKY34 沉默幼苗 （TRV-SlW34） 中的表达与各自的对照植物相比没有显著差异（图 D）。在正常条件下，在 WRKY34 沉默后，幼苗中没有观察到明显的表型变化（图 D）。值得注意的是，含有 ShWRKY34 基因的 LA3942 和 LA1777 的 TRV 幼苗比含有 SlWRKY34 基因的 LA4024 的 TRV 幼苗表现出更强的耐寒性。 较低的相对电解质泄漏 （REL）、较高的光系统 II 最大光化学效率 （Fv/Fm） 和较高的冷应激存活率证明了这一点（图 D）。2b-d 和补充图5a）. 在 LA3942 和 LA1777 中沉默 ShWRKY34 在番茄幼苗中仍然保持了很强的耐寒性。重要的是，在 LA4024 中沉默 SlWRKY34 显着提高了耐冷性，与冷应激下的 TRV 相比，REL 更低，Fv/Fm 更高，存活率更高（图 D）。2b-d 和补充图5a）。

a Expression levels of WRKY34 in introgression line LA3942, its background material LA4024, donor parent LA1777 and their WRKY34-silenced seedlings under cold stress. Total RNA was isolated from leaf samples after 6 h of cold stress. Data are presented as means ± SD of three biological replicates. The relative expression levels of WRKY34 in TRV of LA4024, LA3942 and LA1777 under normal conditions were set to “1”. b Phenotypes of LA3942, LA4024, LA1777 and their WRKY34-silenced seedlings at 7 d after cold treatment. Eight plants of each genotype and treatment were tested with similar results. Bar: 10 cm. Relative electrolyte leakage (REL, c) and maximum photochemical efficiency of photosystem II (Fv/Fm, d) in LA4024, LA3942, LA1777 and their WRKY34-silenced seedlings at 7 d after cold treatment. The false color code depicted at the bottom of the images ranges from 0 (black) to 1 (purple). Bar: 2 cm. Data are presented as means of four biological replicates ± SD (c) or means of eight leaflets from independent plants (d). e Expression levels of CBFs and CORs in LA4024, LA3942, LA1777 and their WRKY34-silenced seedlings under cold stress. Total RNA was isolated from leaf samples after 6 h of cold stress. The relative expression levels of CBFs and CORs in TRV of LA4024, LA3942 and LA1777 under normal conditions were set to “1”. At least twice experiments were repeated independently with similar results. Data are presented as means ± SD of three biological replicates. Different letters indicate significant differences (P < 0.05) according to one-way ANOVA with Duncan’s multiple range test. TRV, non-silenced seedlings; TRV-SlW34, SlWRKY34-silenced seedlings; TRV-ShW34, ShWRKY34-silenced seedlings. Source data are provided as a Source Data file.
a 低温胁迫下 WRKY34 在渗入系 LA3942 、其背景材料 LA4024、供体亲本 LA1777 及其 WRKY34 沉默幼苗中的表达水平。冷应激 6 小时后，从叶片样品中分离总 RNA。数据以 3 个生物学重复± SD 平均值表示。在正常条件下，WRKY34 在 LA4024 、 LA3942 和 LA1777 的 TRV 中的相对表达水平设置为 “1”。b 冷处理后 7 d LA3942、LA4024、LA1777 及其 WRKY34 沉默幼苗的表型。对每种基因型和处理的 8 株植物进行了测试，结果相似。杆：10 厘米。冷处理后 7 d，LA4024、LA3942、LA1777 及其 WRKY34 沉默幼苗的相对电解质泄漏 （REL， c） 和光系统 II 的最大光化学效率 （Fv/Fm， d）。图像底部描绘的假色代码范围从 0（黑色）到 1（紫色）。杆：2 厘米。数据以 SD （c） ± 4 个生物学重复的平均值或来自独立植物的 8 个小叶 （d） 的平均值表示。e 低温胁迫下 LA4024 、 LA3942 、 LA1777 及其 WRKY34 沉默幼苗中 CBFs 和 CORs 的表达水平。冷应激 6 小时后，从叶片样品中分离总 RNA。正常条件下 LA4024 、 LA3942 和 LA1777 在 TRV 中 CBFs 和 CORs 的相对表达水平设置为 “1”。至少独立重复了两次实验，结果相似。数据以 3 个生物学重复± SD 平均值表示。根据 Duncan 多范围检验的单因素方差分析，不同的字母表示显著差异 （P < 0.05）。 TRV，非沉默幼苗;TRV-SlW34， SlWRKY34-沉默幼苗;TRV-ShW34， ShWRKY34 沉默幼苗。源数据作为 源数据 文件提供。

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Furthermore, we assessed the expression levels of cold responsive genes CBFs and CORs in the aforementioned tomato plant lines. As shown in Fig. 2e, under cold stress, cold-induced up-regulation of CBFs and CORs in TRV seedlings of LA3942 and LA1777 was significantly higher than that in TRV seedlings of LA4024. Silencing ShWRKY34 in LA3942 and LA1777 maintained similar expression levels of these cold responsive genes after cold treatment compared to their respective TRV control plants. Importantly, silencing of SlWRKY34 in LA4024 significantly increased the expression of these cold responsive genes after cold treatment compared to its TRV control plants (Fig. 2e). These results indicate that SlWRKY34 does not respond to cold stress and negatively regulates cold tolerance in cultivated tomato, while the expression levels of ShWRKY34 are decreased in both wild tomato and the ShWRKY34 introgression line with strong cold tolerance.
此外，我们评估了上述番茄植株系中冷响应基因 CBFs 和 CORs 的表达水平。如图 1 所示。2e，在低温胁迫下，LA3942 和 LA1777 TRV 幼苗中低温诱导的 CBFs 和 CORs 上调显著高于 LA4024 的 TRV 幼苗。与各自的 TRV 对照植物相比，在 LA3942 和 LA1777 中沉默 ShWRKY34 在冷处理后保持了这些冷响应基因的相似表达水平。重要的是，与其 TRV 对照植物相比，在 LA4024 中沉默 SlWRKY34 在冷处理后显着增加了这些冷反应基因的表达（图 D）。这些结果表明，SlWRKY34 对冷胁迫不响应，负向调节栽培番茄的耐寒性，而 ShWRKY34 在野生番茄和耐寒性强的 ShWRKY34 渗入系中的表达水平均降低。

Cold-suppressed WRKY34 expression is associated with a 60 bp InDel in its promoter region
冷抑制的 WRKY34 表达与其启动子区的 60 bp InDel 相关

To further elucidate the role of WRKY34 in tomato cold tolerance, we compared protein sequences of WRKY34 from six different tomato species (S. lycopersicum; S. lycopersicum var. cerasiforme; S. pimpinellifolium; S. chilense; S. pennellii; S. habrochaites) through amino acid alignments. The comparison revealed that WRKY34 proteins across different tomato species were similar and conserved. For example, there are only ten amino acid differences among the six different tomato species, with merely four amino acid distinctions between cultivated tomato S. lycopersicum and wild tomato S. habrochaites (Supplementary Fig. 6a). We subsequently constructed CaMV 35S promoter-driven SlWRKY34 and ShWRKY34 overexpressing lines (OE), as well as slwrky34 mutants through CRISPR/Cas9-mediated techniques in LA4024 background. Under normal conditions, the wrky34 mutants exhibited smaller fruits and fewer seeds per fruit compared to wild-type (WT) (Supplementary Fig. 7). Moreover, tissue-specific expression analysis revealed that tomato WRKY34 is prominently expressed in roots, followed by flowers and buds, with lower expression levels observed in leaves, and the lowest expression in fruits (Supplementary Fig. 8a). Consistently, WRKY34 protein accumulation was highest in roots and lowest in fruits (Supplementary Fig. 8b). Interestingly, both overexpression of SlWRKY34 and ShWRKY34 compromised seedlings cold tolerance, evidenced by higher REL, lower Fv/Fm and lower survival rate compared to WT (Supplementary Figs. 5b and 6b–d). Conversely, slwrky34 mutants exhibited extreme tolerance to cold stress, displaying lower REL, higher Fv/Fm and higher survival rate than WT seedlings (Supplementary Figs. 5b and 6b–d). Additionally, overexpression of WRKY34 significantly suppressed the expression of cold responsive genes CBFs and CORs under cold stress, whereas the knockout of WRKY34 promoted the expression of these genes under cold stress (Supplementary Fig. 6e). Hence, both SlWRKY34 and ShWRKY34 negatively regulate tomato cold tolerance.
为了进一步阐明 WRKY34 在番茄耐寒性中的作用，我们比较了来自六种不同番茄物种（S. lycopersicum;S. lycopersicum var. cerasiforme;S. pimpinellifolium;智利链球菌;S. pennellii;S. habrochaites）通过氨基酸比对。比较显示，不同番茄品种的 WRKY34 蛋白相似且保守。例如，六种不同的番茄物种之间只有 10 个氨基酸的差异，栽培番茄 S. lycopersicum 和野生番茄 S. habrochaites 之间只有 4 个氨基酸的区别（补充图 D）。随后，我们在 LA4024 背景下通过 CRISPR/Cas9 介导的技术构建了 CaMV 35S 启动子驱动的 SlWRKY34 和 ShWRKY34 过表达系 （OE） 以及 slwrky34 突变体。在正常条件下，与野生型 （WT） 相比，wrky34 突变体表现出更小的果实和更少的每个果实的种子（补充图 D）。7）. 此外，组织特异性表达分析显示，番茄 WRKY34 在根中显著表达，其次是花和芽，在叶中观察到表达水平较低，在果实中表达最低（补充图 D）。一致地，WRKY34 蛋白在根中积累最高，在果实中最低（补充图 D）。有趣的是，SlWRKY34 和 ShWRKY34 的过表达都损害了幼苗的耐寒性，与 WT 相比，更高的 REL、更低的 Fv/Fm 和更低的存活率证明了这一点（补充图5b 和 6b-d）。 相反，slwrky34 突变体表现出对冷胁迫的极强耐受性，表现出比 WT 幼苗更低的 REL、更高的 Fv/Fm 和更高的存活率（补充图5b 和 6b-d）。此外，WRKY34 的过表达在寒冷胁迫下显著抑制了冷反应基因 CBFs 和 CORs 的表达，而 WRKY34 的敲除促进了这些基因在寒冷胁迫下的表达（补充图 1）。因此，SlWRKY34 和 ShWRKY34 均负向调节番茄的耐寒性。

Next, we concluded that differences in expression patterns of WRKY34s under cold stress, rather than their protein function, dictated the disparity in cold tolerance between wild and cultivated tomatoes. To explore the reasons behind the differential expression patterns and chromatin accessibilities of WRKY34 between wild and cultivated tomatoes under cold stress, we amplified and sequenced the 3000 bp length promoter of SlWRKY34 and the 3019 bp length promoter of ShWRKY34 (Supplementary Fig. 9). Evidently, compared to the SlWRKY34 promoter of cultivated tomato, we identified a 60 bp insertion at −2315 bp upstream of the ShWRKY34 translation initiation site (ATG) in wild tomato, precisely situated in the opening chromatin regions of the ShWRKY34 promoter under cold stress in wild tomato LA1777 (Fig. 3a and Supplementary Fig. 9). Two cis-elements, W-box and GATA-box, were identified in the 60 bp InDel by PLANTCARE software (Supplementary Fig. 9). To verify whether the 60 bp InDel of the WRKY34 promoter is accountable for the variation in its expression levels under cold stress, we performed a dual-luciferase (LUC) transcriptional activation assay in tobacco. Tobacco leaves were transformed with constructs containing the LUC reporter gene driven by SlWRKY34 and ShWRKY34 promoters. Under cold stress, the SlWRKY34 promoter (pSlW34) did not obviously affect LUC activity, whereas the ShWRKY34 promoter (pShW34) significantly inhibited the expression of LUC reporter gene. Insertion of the 60 bp InDel at position −2350 in the context of the SlWRKY34 promoter (pSlW34^+60bp) led to a significant decrease in LUC activity after cold stress (Fig. 3b, c). To determine whether two cis-elements are involved in cold-suppressed WRKY34 expression, we mutated W-box and GATA-box in the 60 bp InDel of the ShWRKY34 promoter (pShW34^mW-box and pShW34^mGATA-box) and fused them to LUC reporter constructs, respectively (Fig. 3b). The results revealed that pShW34^mGATA-box restored cold-suppressed LUC activity, whereas pShW34^mW-box still exhibited significant suppression of LUC activity after cold stress (Fig. 3c), suggesting that the cis-element GATA-box within the 60 bp InDel of the ShWRKY34 promoter plays a crucial role in suppressing ShWRKY34 expression under cold stress. Furthermore, we constructed a pSlW34^+30bp/LUC fusion vector, wherein only a 30 bp InDel containing the GATA-box was inserted at position −2350 in the context of the SlWRKY34 promoter, and measured LUC activity. Interestingly, compared with pShW34, pSlW34^+60bp and pShW34^mW-box, the LUC activity of pSlW34^+30bp was only partially reduced under cold stress, suggesting that both the cis-element GATA-box and the entire 60 bp InDel fragment are important for the suppression of WRKY34 transcription after cold stress (Fig. 3c).
接下来，我们得出结论，WRKY34s 在低温胁迫下表达模式的差异，而不是它们的蛋白质功能，决定了野生和栽培番茄之间耐寒性的差异。为了探究低温胁迫下野生和栽培番茄之间 WRKY34 的差异表达模式和染色质可及性背后的原因，我们对 SlWRKY34 的 3000 bp 长度启动子和 ShWRKY34 的 3019 bp 长度启动子进行了扩增和测序（补充图 D）。显然，与栽培番茄的 SlWRKY34 启动子相比，我们在野生番茄中发现了 ShWRKY34 翻译起始位点 （ATG） 上游 −2315 bp 处的 60 bp 插入，正好位于野生番茄 LA1777 低温胁迫下 ShWRKY34 启动子的开放染色质区域（图 D）。图3a和补充图。9）. 通过 PLANTCARE 软件在 60 bp InDel 中鉴定出两个 cis 元件，W-box 和 GATA-box（补充图 D）。9）. 为了验证 WRKY34 启动子的 60 bp InDel 是否负责其在低温胁迫下表达水平的变化，我们在烟草中进行了双荧光素酶 （LUC） 转录激活测定。用含有 SlWRKY34 和 ShWRKY34 启动子驱动的 LUC 报告基因的构建体转化烟叶。在低温胁迫下，SlWRKY34 启动子 （pSlW34） 对 LUC 活性没有明显影响，而 ShWRKY34 启动子 （pShW34） 显著抑制 LUC 报告基因的表达。 在 SlWRKY34 启动子 （pSlW34^+60bp） 的背景下，将 60 bp InDel 插入 −2350 位点导致冷应激后 LUC 活性显著降低（图 D）。3b， c） 的为了确定两个 cis 元件是否参与冷抑制的 WRKY34 表达，我们突变了 ShWRKY34 启动子（pShW34^mW-box 和 pShW34^mGATA-box）的 60 bp InDel 中的 W-box 和 GATA-box，并将它们分别融合到 LUC 报告基因构建体上（图 D）。结果表明，pShW34^mGATA-box 恢复了冷抑制的 LUC 活性，而 pShW34^mW-box 在冷应激后仍然表现出对 LUC 活性的显着抑制（图 D）。3c），表明 ShWRKY34 启动子的 60 bp InDel 内的 c是元件 GATA-box 在低温胁迫下抑制 ShWRKY34 表达中起着至关重要的作用。此外，我们构建了一个 pSlW34^+30bp/LUC 融合载体，其中在 SlWRKY34 启动子的背景下，仅将含有 GATA 盒的 30 bp InDel 插入到 −2350 位，并测量 LUC 活性。 有趣的是，与 pShW34、pSlW34^+60bp 和 pShW34^mW-box 相比，pSlW34^+30bp 的 LUC 活性在低温胁迫下仅部分降低，表明顺式元件 GATA-box 和整个 60 bp InDel 片段对于低温胁迫后抑制 WRKY34 转录都很重要（图 D）。3c）。

a WRKY34 gene structure in cultivated and wild tomatoes. The gray box represents 5’ UTR, the green box represents the coding sequence, the orange box represents the promoter and the black line in the promoter represents the 60 bp InDel region. b Schematic diagram of the reporter constructs with the full-length promoters of WRKY34 or different mutated WRKY34 promoters fused to the LUC reporter gene in the vector pGreenII 0800-LUC, while the internal control REN reporter gene was driven by the CaMV 35S promoter. pSlW34, full-length promoter of SlWRKY34; pShW34, full-length promoter of ShWRKY34; pSlW34^+60bp, SlWRKY34 full-length promoter inserts a 60 bp InDel from ShWRKY34 promoter; pShW34^mW-box, ShWRKY34 full-length promoter mutates W-box in the 60 bp InDel; pShW34^mGATA-box, ShWRKY34 full-length promoter mutates GATA-box in the 60 bp InDel; pSlW34^+30bp, SlWRKY34 full-length promoter inserts a 30 bp InDel from ShWRKY34 promoter. c LUC relative activities driven by different promoters were determined in transient transgenic tobacco plants 6 h after cold stress. The LUC relative activity driven by pSlW34 in transient expressed tobacco leaves under normal conditions was set to “1”. Data are presented as means ± SD of six biological replicates. The experiment was repeated three times with similar results. Different letters indicate significant differences (P < 0.05) according to one-way ANOVA with Duncan’s multiple range test. d The 60 bp InDel of 181 cultivated tomatoes, 74 cherry tomatoes, 58 currant tomatoes and 63 wild tomatoes. e The 60 bp InDel of 63 different wild tomatoes. f The foldchange of WRKY34 expression levels under cold stress in different tomato varieties. The horizontal line in the middle indicates mean. The experiment used three biological replicates in one experiment and was repeated twice with similar results. Source data are provided as a Source Data file.
栽培和野生西红柿中的 WRKY34 基因结构。灰色框代表 5' UTR，绿色框代表编码序列，橙色框代表启动子，启动子中的黑线代表 60 bp InDel 区。b WRKY34 全长启动子或不同突变的 WRKY34 启动子与载体 pGreenII 0800-LUC 中的 LUC 报告基因融合的报告基因构建体示意图，而内部对照 任 报告基因由 CaMV 35S 启动子驱动。pSlW34，SlWRKY34 的全长启动子;pShW34，ShWRKY34 的全长启动子;pSlW34^{+60 bp，SlWRKY34} 全长启动子插入来自 ShWRKY34 启动子的 60 bp InDel; pShW34 ^{mW-box，ShWRKY34} 全长启动子在 60 bp InDel 中突变 W-box; pShW34^{mGATA-box，ShWRKY34} 全长启动子在 60 bp InDel 中突变 GATA-box; pSlW34⁺³⁰ bp，SlWRKY34 全长启动子插入来自 ShWRKY34 启动子的 30 bp InDel。c 在低温胁迫后 6 h 的瞬时转基因烟草植株中测定不同启动子驱动的 LUC 相对活性。在正常条件下，pSlW34 在瞬时表达的烟叶中驱动的 LUC 相对活性设置为 “1”。数据以 6 次生物学重复的 SD ±平均值表示。实验重复 3 次，结果相似。根据 Duncan 多极<范围检验的单因素方差分析，不同的字母表示显著差异 （P 0.05）。 d 181 个栽培番茄、74 个樱桃番茄、58 个醋栗番茄和 63 个野生番茄的 60 bp InDel。e 63 种不同野生西红柿的 60 bp InDel。f 不同番茄品种 WRKY34 表达水平在低温胁迫下的倍数变化。中间的水平线表示平均值。该实验在一次实验中使用了 3 次生物学重复，并重复了两次，结果相似。源数据作为 源数据 文件提供。

Full size image

To further explore whether the 60 bp InDel has been influenced and selected by evolution and domestication, we analyzed the variation of the 60 bp InDel in 181 cultivated tomatoes (S. lycopersicum), 74 cherry tomatoes (S. lycopersicum var. cerasiforme), 58 currant tomatoes (S. pimpinellifolium) and 63 wild tomatoes including 3 S. cheesmaniae, 2 S. galapagense, 8 S. arcanum, 3 S. chmielewskii, 9 S. neorickii, 3 S. huaylasense, 4 S. corneliomulleri, 4 S. peruvianum, 12 S. chilense, 10 S. habrochaites, 4 S. pennellii and 1 S. sitiens accessions (Supplementary Data 6). Surprisingly, we observed no 60 bp insertion in WRKY34 promoters across all cultivated tomatoes, cherry tomatoes, currant tomatoes, and two wild tomato species, S. cheesmaniae and S. galapagense accessions. However, the 60 bp insertions were prevalent in 91.4% (53 out of 58) of other wild tomatoes, including all S. chmielewskii, S. neorickii, S. corneliomulleri, S. peruvianum, S. habrochaites, S. pennellii, and S. sitiens accessions, as well as 87.5% (7 out of 8) S. arcanum, 75% (9 out of 12) S. chilense, and 66.7% (2 out of 3) S. huaylasense accessions (Fig. 3d, e and Supplementary Data 6). To verify the relationship between the 60 bp InDel and WRKY34 expression in response to cold stress, we selected a subset of cultivated tomatoes, cherry tomatoes, currant tomatoes and all wild tomatoes to measure the expression levels of WRKY34 at 6 h after cold treatment. The results showed that the WRKY34 variants harboring the 60 bp deletion exhibited no significant alteration in expression after cold stress. In contrast, the expression of WRKY34 variants with the 60 bp insertion demonstrated a notable decrease after cold stress (Fig. 3f and Supplementary Fig. 10). These results suggest that the 60 bp InDel is significantly associated with cold-suppressed WRKY34 expression in all wild and cultivated tomatoes, and the 60 bp insertion in the WRKY34 promoter is prevalent in wild tomatoes, yet it has progressively vanished during the extended evolutionary transition to currant tomatoes.
为了进一步探讨 60 bp InDel 是否受到进化和驯化的影响和选择，我们分析了 181 个栽培番茄 （S. lycopersicum）、74 个樱桃番茄 （S. lycopersicum var. cerasiforme）、58 个醋栗番茄 （S. pimpinellifolium） 和 63 个野生番茄（包括 3 个 S. cheesmaniae、2 个 S. galapagense、8 个 S. arcanum、 3 个 S. chmielewskii， 9 个 S. neorickii， 3 个 S. huaylasense， 4 个 S. corneliomulleri， 4 个 S. peruvianum， 12 个 S. chilense， 10 个 S. habrochaites， 4 个 S. pennellii 和 1 个 S. sitiens 种质（补充数据 6）。令人惊讶的是，我们观察到在所有栽培番茄、樱桃番茄、醋栗番茄和两种野生番茄物种 S. cheesmaniae 和 S. galapagense 种质中，WRKY34 启动子中没有 60 bp 的插入。然而，60 bp 插入在 91.4%（58 个中的 53 个）的其他野生西红柿中普遍存在，包括所有 S. chmielewskii、S. neorickii、S. corneliomulleri、S. peruvianum、S. habrochaites、S. pennellii 和 S. sitiens，以及 87.5%（8 个中的 7 个）S. arcanum、75%（12 个 9 个）智利 S. chilense 和 66.7%（3 个 2 个）Huaylasense 沙门柩种质（图 D）。3d、e 和补充数据 6）。为了验证 60 bp InDel 和 WRKY34 响应低温胁迫表达之间的关系，我们选择了栽培番茄、樱桃番茄、醋栗番茄和所有野生番茄的一个子集来测量冷处理后 6 h WRKY34 的表达水平。 结果表明，携带 60 bp 缺失的 WRKY34 变体在冷应激后表达没有显着改变。相比之下，插入 60 bp 的 WRKY34 变体的表达在冷应激后表现出显着降低（图 D）。3f 和补充图这些结果表明，60 bp InDel 与所有野生和栽培番茄中冷抑制的 WRKY34 表达显著相关，WRKY34 启动子中的 60 bp 插入在野生番茄中普遍存在，但在向醋栗番茄的延长进化过渡过程中，它已逐渐消失。

SWIBs and GATA29 directly bind to the 60 bp InDel fragment of the WRKY34 promoter
SWIB 和 GATA29 直接与 WRKY34 启动子的 60 bp InDel 片段结合

To explore how the 60 bp InDel fragment represses WRKY34 expression, we employed a yeast one-hybrid (Y1H) screen using the 60 bp InDel region as bait DNA. Prey proteins from a tomato complementary DNA library, fused with the yeast GAL4 transcription activation domain (GAL4 AD), were screened. Out of 297 putative DNA-binding proteins identified, we chose a GATA family transcription factor, SlGATA29 capable of binding to the GATA-box, and two SWIB/MDM2 domain proteins, SlSWIBa and SlSWIBb, known to influence chromatin opening (Supplementary Data 7), for further analysis.
为了探索 60 bp InDel 片段如何抑制 WRKY34 表达，我们采用了酵母单杂交 （Y1H） 筛选，使用 60 bp InDel 区域作为诱饵 DNA。筛选来自番茄互补 DNA 文库的猎物蛋白，与酵母 GAL4 转录激活结构域 （GAL4 AD） 融合。在鉴定的 297 种推定的 DNA 结合蛋白中，我们选择了一个 GATA 家族转录因子，能够与 GATA 盒结合的 SlGATA29，以及两个已知影响染色质开放的 SWIB/MDM2 结构域蛋白 SlSWIBa 和 SlSWIBb（补充数据 7），用于进一步分析。

Using full-sequence constructs of these genes, we performed gene-specific Y1H assays to determine their specific binding to the aforementioned 60 bp InDel. Yeast cells containing the 60 bp bait vector and either the pGADT7-SlGATA29 vector or pGADT7-SlSWIBa/b vectors grew on the SD-Leu media with 150 ng ml⁻¹ aureobasidin A (AbA) (SD-Leu¹⁵⁰). Conversely, transformants lacking SlGATA29 or SlSWIBa/b failed to grow on this media (Fig. 4a). To identify the core DNA binding sites of SWIB/MDM2 domain proteins SlSWIBa/b, we created six different 60 bp mutation probes with various mutation sites (mu1-mu6) and performed electrophoretic mobility shift assay (EMSA) and microscale thermophoresis (MST). The results revealed mu4 (TGATAA) as the common core DNA binding site of SlSWIBa and SlSWIBb, consistent with GATA-box; hence, we named it as SWIB-mu4 (Supplementary Fig. 11). Transformants with mutated GATA-box or SWIB-mu4 (mut^GATA-box or mut^SWIB-mu4) bait vectors did not grow on SD-Leu¹⁵⁰ media (Fig. 4a), indicating that SlGATA29 and SlSWIBa/b could bind to the GATA-box and SWIB-mu4 of the 60 bp InDel in yeast, respectively. EMSA results further confirmed that SlGATA29 and SlSWIBa/b bound to the GATA-box and SWIB-mu4 in the 60 bp InDel, respectively (Fig. 4b). However, SlSWIBa/b could not bind to a 30 bp probe with a complete SWIB-mu4, indicating that the binding of SlSWIBa/b to the 60 bp InDel requires not only the SWIB-mu4 but also the full-length 60 bp DNA fragment (Fig. 4b). Previous studies have primarily considered SWIB/MDM2 domain proteins as regulators of chromatin accessibility through their influence on nucleosomes³³. To date, there has been no report on the direct binding of SWIB/MDM2 proteins to chromatin DNA. To further identify the key amino acid sites in SWIBs for binding to the 60 bp InDel, we made an accurate prediction of protein-nucleic acid complexes using RoseTTAFoldNA³⁴. Predicted results suggested that three conserved amino acids in two SlSWIB homologous proteins, including Arg6 in both SlSWIBa and SlSWIBb, Leu46 in SlSWIBa and Leu44 in SlSWIBb, and Lys88 in SlSWIBa and Lys86 in SlSWIBb, might be involved in binding to the 60 bp InDel through hydrogen bonds (Fig. 4c). To verify the key binding roles of these amino acids, we separately mutated these three amino acids and also mutated all three amino acids together, then tested their binding with the 60 bp InDel. Both MST and Y1H results showed that mutating any single amino acid still allowed SlSWIBa/b to bind to the 60 bp InDel, but when all three amino acids were mutated to alanine, they no longer bound to the 60 bp InDel in vitro (Fig. 4d, e). These results further indicate that SWIBs have the ability to directly bind to DNA, and we infer that they would form a helical protein to wrap around DNA, with SWIB-mu4 being the key recognition or binding site.
使用这些基因的全序列构建体，我们进行了基因特异性 Y1H 测定，以确定它们与上述 60 bp InDel 的特异性结合。含有 60 bp 诱饵载体和 pGADT7-SlGATA29 载体或 pGADT7-SlSWIBa/b 载体的酵母细胞在含有 150 ng ^ml-1 金霉素 A （AbA） （SD-Leu¹⁵⁰） 的 SD-Leu 培养基上生长。相反，缺乏 SlGATA29 或 SlSWIBa/b 的转化体无法在该培养基上生长（图 D）。为了鉴定 SWIB/MDM2 结构域蛋白 SlSWIBa/b 的核心 DNA 结合位点，我们创建了六种不同的 60 bp 突变探针，具有不同突变位点 （mu1-mu6），并进行了电泳迁移率变化测定 （EMSA） 和微量热泳 （MST）。结果显示 mu4 （TGATAA） 是 SlSWIBa 和 SlSWIBb 的共同核心 DNA 结合位点，与 GATA-box 一致;因此，我们将其命名为 SWIB-mu4（补充图 1）。11）. 具有突变的 GATA-box 或 SWIB-mu4 （mut^GATA-box 或 mut^SWIB-mu4） 诱饵载体的转化体在 SD-Leu¹⁵⁰ 培养基上没有生长（图 D）。4a），表明 SlGATA29 和 SlSWIBa/b 可以分别与酵母中 60 bp InDel 的 GATA-box 和 SWIB-mu4 结合。EMSA 结果进一步证实，SlGATA29 和 SlSWIBa/b 分别与 60 bp InDel 中的 GATA-box 和 SWIB-mu4 结合（图 D）。然而，SlSWIBa/b 不能与具有完整 SWIB-mu4 的 30 bp 探针结合，这表明 SlSWIBa/b 与 60 bp InDel 的结合不仅需要 SWIB-mu4，还需要全长 60 bp DNA 片段（图 D）。以前的研究主要认为 SWIB/MDM2 结构域蛋白通过它们对核小体的影响作为染色质可及性的调节因子³³。 迄今为止，还没有关于 SWIB/MDM2 蛋白与染色质 DNA 直接结合的报道。为了进一步鉴定 SWIBs 中与 60 bp InDel 结合的关键氨基酸位点，我们使用 RoseTTAFoldNA³⁴ 对蛋白质-核酸复合物进行了准确预测。预测结果表明，两种 SlSWIB 同源蛋白中的三个保守氨基酸，包括 SlSWIBa 和 SlSWIBb 中的 Arg6、SlSWIBa 中的 Leu46 和 SlSWIBb 中的 Leu44，以及 SlSWIBa 中的 Lys88 和 SlSWIBb 中的 Lys86，可能参与通过氢键与 60 bp InDel 结合（图 D）。为了验证这些氨基酸的关键结合作用，我们分别突变了这三种氨基酸，并将所有三种氨基酸一起突变，然后测试了它们与 60 bp InDel 的结合。MST 和 Y1H 结果表明，突变任何单个氨基酸仍然允许 SlSWIBa/b 与 60 bp 的 InDel 结合，但是当所有三个氨基酸都突变为丙氨酸时，它们在体外不再与 60 bp 的 InDel 结合（图 D）。4d， e）。这些结果进一步表明 SWIBs 具有直接与 DNA 结合的能力，我们推断它们会形成螺旋蛋白包裹 DNA，其中 SWIB-mu4 是关键的识别或结合位点。

a Yeast one-hybrid assay (Y1H) testing the binding of SlGATA29 and SlSWIBa/b to pAbAi-60bp in the SD-Leu medium with or without 150 ng ml^-1 AbA. Empty vector containing the AD serves as a negative control. b EMSA results showing SlGATA29 and SlSWIBa/b directly bind to the 60 bp InDel. Probe sequences are shown (top). mu^GATA-box and mu^SWIB-mu4, mutated probes in which the TGATAA motif was changed to AAAAAA. c Complex structures of SlSWIBa with the 60 bp DNA fragment (upper panel) and SlSWIBb with the 60 bp DNA fragment (lower panel). Blue and red indicate the SWIB/MDM2 domain and the predicted binding site, respectively. SWIB-mu4 is highlighted in red. d MST assays of the binding of SlSWIBa/b and their three amino acid mutations to the 60 bp InDel. Kd, dissociation constant. e Y1H testing the binding of SlSWIBa/b and their three amino acid mutations to pAbAi-60bp in the SD-Leu medium with or without 150 ng ml⁻¹ AbA. f Transient dual-luciferase (dual-LUC) assay in tobacco leaves. Empty vector (EV) was included as control. Data are presented as means ± SD of six biological replicates. ChIP-qPCR assays showing the binding of SlGATA29 (g) and SlSWIBb (h) to the 60 bp InDel in vivo. Data are presented as means ± SD of three biological replicates. i Expression levels of WRKY34 in LA4024, LA3942 and SlGATA29 overexpressing (SlGATA29-OE) seedlings and SlSWIBb overexpressing (SlSWIBb-OE) seedlings in the background of LA4024 or LA3942 under cold stress. Total RNA was isolated from leaf samples after 6 h of cold stress. Data are presented as means ± SD of three biological replicates. The relative expression levels of WRKY34 in WT of LA4024 and LA3942 under normal conditions were set to “1”. Experiments in (a, b, d, e) were repeated twice, and in (f–i), three times, all yielding similar results. Different letters above bars indicate significant differences (P < 0.05) determined using one-way ANOVA with Duncan’s multiple range test. Source data are provided as a Source Data file.
酵母单杂交测定 （Y1H） 在含或不含 150 ng ^ml-1 AbA 的 SD-Leu 培养基中检测 SlGATA29 和 SlSWIBa/b 与 pAbAi-60bp 的结合。含有 AD 的空载体用作阴性对照。b EMSA 结果显示 SlGATA29 和 SlSWIBa/b 直接与 60 bp InDel 结合。显示探针序列（顶部）。mu^GATA-box 和 mu^SWIB-mu4，突变探针，其中 TGATAA 基序变为 AAAAAA。c 具有 60 bp DNA 片段的 SlSWIBa 的复杂结构（上图）和具有 60 bp DNA 片段的 SlSWIBb（下图）。蓝色和红色分别表示 SWIB/MDM2 结构域和预测的结合位点。SWIB-mu4 以红色突出显示。d SlSWIBa/b 及其三个氨基酸突变与 60 bp InDel 结合的 MST 测定。Kd，解离常数。e Y1H 在有或没有 150 ng ^ml-1 AbA 的 SD-Leu 培养基中检测 SlSWIBa/b 及其三个氨基酸突变与 pAbAi-60bp 的结合。f 烟叶中的瞬时双荧光素酶 （dual-LUC） 测定。空向量 （EV） 作为对照。数据以 6 次生物学重复的 SD ±平均值表示。ChIP-qPCR 检测显示 SlGATA29 （g） 和 SlSWIBb （h） 在体内与 60 bp InDel 的结合。数据以 3 个生物学重复± SD 平均值表示。i 在低温胁迫下，LA4024、LA3942 和 SlGATA29 过表达 （SlGATA29-OE） 幼苗以及 LA4024 或 LA3942 背景下 SlSWIBb 过表达 （SlSWIBb-OE） 幼苗中 WRKY34 的表达水平。冷应激 6 小时后，从叶片样品中分离总 RNA。数据以 3 个生物学重复± SD 平均值表示。 在正常条件下，LA4024 和 LA3942 在 WT 中 WRKY34 的相对表达水平设置为 “1”。（a， b， d， e） 中的实验重复两次，（f-i） 中的实验重复 3 次，均产生相似的结果。条形上方的不同字母表示使用单因素方差分析和 Duncan 多范围检验确定的显著差异 （P < 0.05）。源数据作为 源数据 文件提供。

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To investigate the effect of SlGATA29 and SlSWIBa/b on downstream WRKY34 gene expression, we performed dual-LUC assays and found that SlGATA29 suppressed LUC activity when WRKY34 promoters contained the GATA-box (pShW34, pSlW34^+60bp, pShW34^mW-box and pSlW34^+30bp), but had little effect on WRKY34 promoters without the GATA-box (pSlW34 and pShW34^mGATA-box). Interestingly, when the reporter gene was co-transfected with SlSWIBa or SlSWIBb gene, the LUC activity of all WRKY34 promoters did not change, suggesting that SlSWIBs do not directly regulate WRKY34 expression (Fig. 4f). To verify the binding of SlGATA29 and SlSWIBs to the 60 bp InDel in vivo, we overexpressed SlGATA29 and SlSWIBb in cultivated tomato LA4024 and introgression line LA3942, respectively. ChIP-qPCR analysis showed that SlGATA29 and SlSWIBb could not bind to the SlWRKY34 promoter of LA4024 with or without cold treatment, due to the absence of the 60 bp InDel. SlGATA29 could not bind to the ShWRKY34 promoter of LA3942 under normal conditions, but could bind to its promoter under cold stress (Fig. 4g). At normal temperature, SlSWIBb could directly bind to the ShWRKY34 promoter of LA3942, and the binding was further increased after cold stress (Fig. 4h). Moreover, overexpression of SlGATA29 or SlSWIBb in LA3942 background suppressed the expression of ShWRKY34 under cold stress, while the expression of SlWRKY34 in LA4024 background did not respond to cold stress (Fig. 4i). Additionally, we compared the protein sequences of GATA29 and SWIBs between cultivated tomato S. lycopersicum and wild tomato S. habrochaites. The comparison results showed only one amino acid difference between SlGATA29 and ShGATA29 (Supplementary Fig. 12a). Compared with two homologous SlSWIBa and SlSWIBb, only one ShSWIB was identified in S. habrochaites LA1777. The protein sequence homology of SlSWIBa and SlSWIBb was 68.6%, and the protein sequence homology of ShSWIB and SlSWIBb was 99.2% with only one amino acid difference (Supplementary Fig. 12b). Y1H and EMSA results also demonstrated that ShGATA29 and ShSWIB directly bind the 60 bp InDel of WRKY34 promoter by specifically interacting with GATA-box and SWIB-mu4, respectively. Additionally, the binding of ShSWIB to the 60 bp InDel also requires a full-length 60 bp DNA (Supplementary Fig. 12c, d). Moreover, dual-LUC assays indicated that ShGATA29 suppressed LUC activity when WRKY34 promoters contained the GATA-box (pShW34, pSlW34^+60bp, pShW34^mW-box and pSlW34^+30bp), but had little effect on WRKY34 promoters lacking the GATA-box (pSlW34 and pShW34^mGATA-box). However, the LUC activity derived from all WRKY34 promoters did not exhibit LUC suppression when the reporter was co-transfected with ShSWIB (Supplementary Fig. 12e). These findings suggest that both the transcription factor GATA29 and the SWIB/MDM2 domain protein SWIBs, found in both wild and cultivated tomatoes, can bind to the 60 bp InDel fragment of the WRKY34 promoter.
为了研究 SlGATA29 和 SlSWIBa/b 对下游 WRKY34 基因表达的影响，我们进行了双 LUC 测定，发现当 WRKY34 启动子包含 GATA-box （pShW34、pSlW34+^60bp、pShW34^mW-box 和 pSlW34^+30bp） 时，SlGATA29 抑制了 LUC 活性，但对没有 GATA-box 的 WRKY34 启动子几乎没有影响 （pSlW34 和pShW34^mGATA-box）。有趣的是，当报告基因与 SlSWIBa 或 SlSWIBb 基因共转染时，所有 WRKY34 启动子的 LUC 活性都没有改变，这表明 SlSWIBs 不直接调节 WRKY34 表达（图 D）。为了验证 SlGATA29 和 SlSWIBs 在体内与 60 bp InDel 的结合，我们分别在培养的番茄 LA4024 和渗入系 LA3942 中过表达 SlGATA29 和 SlSWIBb。ChIP-qPCR 分析显示，由于缺乏 60 bp InDel，无论是否冷处理，SlGATA29 和 SlSWIBb 都不能与 LA4024 的 SlWRKY34 启动子结合。SlGATA29 在正常条件下不能与 LA3942 的 ShWRKY34 启动子结合，但在冷应激下可以与其启动子结合（图 D）。4g）。在常温下，SlSWIBb 可以直接与 LA3942 的 ShWRKY34 启动子结合，并且在冷应激后结合进一步增加（图 D）。此外，在 LA3942 背景下过表达 SlGATA29 或 SlSWIBb 抑制了冷胁迫下 ShWRKY34 的表达，而 SlWRKY34 在 LA4024 背景下的表达对冷胁迫没有响应（图 D）。此外，我们比较了栽培番茄 S. lycopersicum 和野生番茄 S. habrochaites 之间 GATA29 和 SWIBs 的蛋白质序列。比较结果显示 SlGATA29 和 ShGATA29 之间只有一个氨基酸差异（补充图 D）。与两个同源的 SlSWIBa 和 SlSWIBb 相比，在 S. habrochaites LA1777 中仅鉴定出一个 ShSWIB。SlSWIBa 和 SlSWIBb 的蛋白质序列同源性为 68.6%，ShSWIB 和 SlSWIBb 的蛋白质序列同源性为 99.2%，只有一个氨基酸差异（补充图 D）。Y1H 和 EMSA 结果还表明，ShGATA29 和 ShSWIB 分别通过与 GATA-box 和 SWIB-mu4 特异性相互作用直接结合 WRKY34 启动子的 60 bp InDel。此外，ShSWIB 与 60 bp InDel 的结合也需要一个全长 60 bp 的 DNA（补充图 D）。12c， d）。此外，双 LUC 测定表明，当 WRKY34 启动子包含 GATA 盒 （pShW34、pSlW34^+60bp、pShW34^mW-box 和 pSlW34^+30bp） 时，ShGATA29 抑制 LUC 活性，但对缺乏 GATA 盒的 WRKY34 启动子 （pSlW34 和 pShW34^mGATA-box） 影响不大。然而，当报告基因与 ShSWIB 共转染时，来自所有 WRKY34 启动子的 LUC 活性并未表现出 LUC 抑制（补充图 D）。12e）。 这些发现表明，在野生和培养番茄中发现的转录因子 GATA29 和 SWIB/MDM2 结构域蛋白 SWIBs 都可以与 WRKY34 启动子的 60 bp InDel 片段结合。

SWIB and GATA29 synergistically suppress WRKY34 expression through the 60 bp InDel fragment
SWIB 和 GATA29 通过 60 bp InDel 片段协同抑制 WRKY34 的表达

Chromatin remodeling factors are typically recruited to target genes via specific transcription factors, thereby synergistically regulating the expression of target genes³⁵. Interestingly, using yeast two-hybrid (Y2H) assays, we found that SlGATA29 interacted with both SlSWIBa and SlSWIBb in yeast (Fig. 5a). To further validate this interaction, we performed a glutathione S-transferase (GST) pull-down assay. The results revealed that SlGATA29-GST successfully pulled down SlSWIBa-His or SlSWIBb-His, while the negative control GST failed to do so (Fig. 5b). Similarly, a bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of SlGATA29 with SlSWIBa and SlSWIBb in the nucleus (Fig. 5c). Furthermore, the interaction between SlGATA29 and SlSWIBs was verified by co-immunoprecipitation (Co-IP) assays in tobacco leaves through co-expressing SlGATA29-HA and SlSWIBa-GFP or SlGATA29-HA and SlSWIBb-GFP (Fig. 5d). These results indicate that SlGATA29 interacts with both SlSWIBa and SlSWIBb in vitro and in vivo.
染色质重塑因子通常通过特异性转录因子募集到靶基因，从而协同调节靶基因的表达³⁵。有趣的是，使用酵母双杂交 （Y2H） 测定，我们发现 SlGATA29 与酵母中的 SlSWIBa 和 SlSWIBb 相互作用（图 D）。为了进一步验证这种相互作用，我们进行了谷胱甘肽 S-转移酶 （GST） 沉降测定。结果显示，SlGATA29-GST 成功拉低了 SlSWIBa-His 或 SlSWIBb-His，而阴性对照 GST 则未能做到这一点（图 D）。同样，双分子荧光互补 （BiFC） 测定证实了 SlGATA29 与细胞核中 SlSWIBa 和 SlSWIBb 的相互作用（图 2）。此外，通过共表达 SlGATA29-HA 和 SlSWIBa-GFP 或 SlGATA29-HA 和 SlSWIBb-GFP，通过免疫共沉淀 （Co-IP） 测定在烟叶中验证 SlGATA29 和 SlSWIBs之间的相互作用 （图 .这些结果表明 SlGATA29 在体外和体内与 SlSWIBa 和 SlSWIBb 相互作用。

a Yeast two-hybrid assay (Y2H) testing the interactions of SlGATA29 and SlSWIBa/b in yeast. b Pull-down assays show that GST-tagged SlGATA29 physically interacts with His-tagged SlSWIBa or His-tagged SlSWIBb. The green triangle indicates the target band of SlSWIBa-His or SlSWIBb-His pulled down by SlGATA29-GST. The combination of GST and SlSWIBa-His or SlSWIBb-His was used as a negative control. c Bimolecular fluorescence complementation assay (BiFC) showing the interactions between SlGATA29 and SlSWIBa/b in tobacco. Bar: 50 μm. d Co-IP showing the interactions between SlGATA29 and SlSWIBa/b. The green triangle indicates the SlGATA29-HA target band immunoprecipitated with SlSWIBa-GFP or SlSWIBb-GFP. The combination of GFP and SlGATA29-HA was used as a negative control. e Transient dual-luciferase (dual-LUC) assay in tobacco leaves. Empty vector (EV) was included as control. Data are presented as means ± SD of six biological replicates. f ChIP-qPCR assays show that silencing SlSWIBab affects the binding of SlGATA29 to the 60 bp InDel in vivo. Data are presented as means ± SD of three biological replicates. g Expression levels of WRKY34 in SlGATA29 overexpressing lines of LA4024 or LA3942 background and their SlSWIBab-silenced seedlings under cold stress. Total RNA was isolated from leaf samples after 6 h of cold stress. Data are presented as means ± SD of three biological replicates. The relative expression levels of WRKY34 in TRV of SlGATA29 overexpressing lines of LA4024 or LA3942 background under normal conditions were set to “1”. In (a–d), experiments were repeated twice with similar results. In (e–g), experiments were repeated three times with similar results. Different letters above bars indicate significant differences (P < 0.05) determined using one-way ANOVA with Duncan’s multiple range test. TRV, non-silenced seedlings; TRV-SlSWIBab, SlSWIBa and SlSWIBb co-silenced seedlings; SlGATA29-OE/LA4024, SlGATA29 overexpressing lines in LA4024 background; SlGATA29-OE/LA3942, SlGATA29 overexpressing lines in LA3942 background. Source data are provided as a Source Data file.
a 酵母双杂交测定 （Y2H） 测试 SlGATA29 和 SlSWIBa/b 在酵母中的相互作用。b Pull-down 测定显示 GST 标记的 SlGATA29 与 His 标记的 SlSWIBa 或 His 标记的 SlSWIBb 发生物理相互作用。绿色三角形表示 SlSWIBa-His 或 SlSWIBb-His 被 SlGATA29-GST 拉低的目标条带。GST 和 SlSWIBa-His 或 SlSWIBb-His 的组合用作阴性对照。c 双分子荧光互补试验 （BiFC） 显示烟草中 SlGATA29 和 SlSWIBa/b 之间的相互作用。棒：50 μm。d Co-IP 显示 SlGATA29 和 SlSWIBa/b 之间的相互作用。绿色三角形表示用 SlSWIBa-GFP 或 SlSWIBb-GFP 免疫沉淀的 SlGATA29-HA 靶条带。GFP 和 SlGATA29-HA 的组合用作阴性对照。e 烟叶中的瞬时双荧光素酶 （dual-LUC） 测定。空向量 （EV） 作为对照。数据以 6 次生物学重复的 SD ±平均值表示。f ChIP-qPCR 检测显示，沉默 SlSWIBab 会影响体内 SlGATA29 与 60 bp InDel 的结合。数据以 3 个生物学重复± SD 平均值表示。g 寒冷胁迫下过表达 LA4024 或 LA3942 背景的 SlGATA29 细胞系及其 SlSWIBab 沉默幼苗中 WRKY34 的表达水平。冷应激 6 小时后，从叶片样品中分离总 RNA。数据以 3 个生物学重复± SD 平均值表示。在正常条件下，过表达 LA4024 或 LA3942 背景谱系的 SlGATA29 TRV 中 WRKY34 的相对表达水平设置为 “1”。在 （a-d） 中，实验重复两次，结果相似。 在 （e-g） 中，实验重复 3 次，结果相似。条形上方的不同字母表示使用单因素方差分析和 Duncan 多范围检验确定的显著差异 （P < 0.05）。TRV，非沉默幼苗;TRV-SlSWIBab、SlSWIBa 和 SlSWIBb 共沉默幼苗;SlGATA29-OE/LA4024、SlGATA29 在 LA4024 背景下过表达谱系;SlGATA29-OE/LA3942、SlGATA29 在 LA3942 背景下过表达谱系。源数据作为 源数据 文件提供。

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To investigate the effect of the interaction between SlGATA29 and SlSWIBs on downstream WRKY34 gene expression, we performed transient dual-LUC assays with different types of WRKY34 promoters. LUC activity derived from WRKY34 promoters containing the GATA-box (pShW34, pSlW34^+60bp, pShW34^mW-box and pSlW34^+30bp) decreased significantly when co-transfected with SlGATA29, and this reduction was further augmented after co-transfection with SlSWIBa or SlSWIBb, except for pSlW34^+30bp. Conversely, LUC activity derived from WRKY34 promoters lacking the GATA-box (pSlW34 and pShW34^mGATA-box) showed no change when co-transfected with either SlGATA29 alone or with both SlGATA29 and SlSWIBs (Fig. 5e).
为了研究 SlGATA29 和 SlSWIBs 之间相互作用对下游 WRKY34 基因表达的影响，我们使用不同类型的 WRKY34 启动子进行了瞬时双 LUC 测定。当与 SlGATA29 共转染时，含有 GATA-box 的 WRKY34 启动子 （pShW34、pSlW34^+60bp、pShW34^mW-box 和 pSlW34^+30bp） 衍生的 LUC 活性显著降低，与 SlSWIBa 或 SlSWIBb 共转染后，除 pSlW34^+30bp 外，这种降低进一步增强。相反，当单独转染 SlGATA29 或同时转染 SlGATA29 和 SlSWIB 时，来自缺乏 GATA 盒的 WRKY34 启动子（pSlW34 和 pShW34^mGATA-box）的 LUC 活性显示没有变化（图 D）。5e）。

To further examine the synergistic effects of SlGATA29 and SlSWIBs, we performed EMSA in vitro. As shown in Supplementary Fig. 13, while SlGATA29 could specifically bind to 60 bp probe containing the GATA-box, the addition of purified SlSWIBa or SlSWIBb proteins did not significantly affect its binding ability. Therefore, we speculated that the synergistic suppression effect of SlGATA29 and SlSWIBa/b on WRKY34 expression might be linked to chromatin accessibility, and such changes in chromatin accessibility require a eukaryotic environment. As shown in Fig. 5f, in LA4024 where the SlWRKY34 promoter lacks the 60 bp InDel, there was no binding of SlGATA29 to the SlWRKY34 promoter in both TRV and SlSWIBa and SlSWIBb co-silenced seedlings (TRV-SlSWIBab) of SlGATA29 overexpressing lines. Conversely, in LA3942 background, where ShWRKY34 promoter contains the 60 bp InDel fragment, significant binding of SlGATA29 to the ShWRKY34 promoter was observed in TRV of SlGATA29 overexpressing lines. However, when SlSWIBa and SlSWIBb were co-silenced, the binding of SlGATA29 to the ShWRKY34 promoter containing the 60 bp InDel fragment was significantly reduced (Fig. 5f). Correspondingly, the expression of SlWRKY34 in LA4024 background remained unchanged, whereas the expression of ShWRKY34 in SlGATA29 overexpressing lines of LA3942 background was significantly decreased under cold stress. Silencing SlSWIBab in SlGATA29 overexpressing lines of LA3942 background significantly alleviated the decrease in WRKY34 expression under cold stress (Fig. 5g). These results suggest that SlSWIBs can enhance the suppression effect of SlGATA29 on WRKY34 expression, and a complete 60 bp InDel in the WRKY34 promoter is indispensable for achieving the synergistic suppression effect of SlGATA29 and SlSWIBs.
为了进一步检查 SlGATA29 和 SlSWIBs 的协同作用，我们在体外进行了 EMSA。如补充图 1 所示。13，虽然 SlGATA29 可以与含有 GATA-box 的 60 bp 探针特异性结合，但添加纯化的 SlSWIBa 或 SlSWIBb 蛋白并未显着影响其结合能力。因此，我们推测 SlGATA29 和 SlSWIBa/b 对 WRKY34 表达的协同抑制作用可能与染色质可及性有关，而染色质可及性的这种变化需要真核环境。如图 1 所示。5f，在 LA4024 中，SlWRKY34 启动子缺乏 60 bp InDel，在 TRV 和 SlSWIBa 中都没有 SlGATA29 与 SlWRKY34 启动子的结合，而 SlSWIBb 共沉默幼苗 （TRV-SlSWIBab） 过表达系。相反，在 LA3942 背景中，其中 ShWRKY34 启动子包含 60 bp 的 InDel 片段，在 SlGATA29 过表达细胞系的 TRV 中观察到 SlGATA29 与 ShWRKY34 启动子的显着结合。然而，当 SlSWIBa 和 SlSWIBb 共沉默时，SlGATA29 与含有 60 bp InDel 片段的 ShWRKY34 启动子的结合显着降低（图 D）。相应地，SlWRKY34 在 LA4024 背景下的表达保持不变，而 ShWRKY34 在 LA3942 背景下过表达系的 SlGATA29 中的表达在低温胁迫下显著降低。在过表达 LA3942 背景的 SlGATA29 细胞系中沉默 SlSWIBab 可显著缓解低温胁迫下 WRKY34 表达的降低（图 D）。5g）。 这些结果表明，SlSWIBs 可以增强 SlGATA29 对 WRKY34 表达的抑制作用，WRKY34 启动子中完整的 60 bp InDel 对于实现 SlGATA29 和 SlSWIBs 的协同抑制作用是必不可少的。

Given the high protein sequence similarity between SlGATA29 and ShGATA29, as well as between SlSWIBs and ShSWIB, Y2H also detected the interaction between ShGATA29 and ShSWIB in yeast (Supplementary Fig. 14a). We further verified the interaction of ShGATA29 and ShSWIB by performing pull-down assays in vitro (Supplementary Fig. 14b). BiFC and Co-IP results also confirmed the interaction between ShGATA29 and ShSWIB in vivo (Supplementary Fig. 14c, d). Similarly, we performed dual-LUC assays to investigate the synergistic suppression effect of ShGATA29 and ShSWIB (Supplementary Fig. 14e). The results showed that ShSWIB and ShGATA29 exhibit similar functions with SlSWIBs and SlGATA29 in synergistic suppression of WRKY34 expression.
鉴于 SlGATA29 和 ShGATA29 之间以及 SlSWIB 和 ShSWIB 之间的蛋白质序列高度相似性，Y2H 还检测到酵母中 ShGATA29 和 ShSWIB 之间的相互作用（补充图 D）。我们通过在体外进行沉降测定进一步验证了 ShGATA29 和 ShSWIB 的相互作用（补充图 D）。BiFC 和 Co-IP 结果也证实了 ShGATA29 和 ShSWIB 在体内的相互作用（补充图 D）。14c， d）。同样，我们进行了双 LUC 测定以研究 ShGATA29 和 ShSWIB 的协同抑制作用（补充图 D）。结果表明，ShSWIB 和 ShGATA29 在协同抑制 WRKY34 表达方面表现出与 SlSWIBs 和 SlGATA29 相似的功能。

Impact of SWlB and GATA29 on chromatin accessibility and cold tolerance via the 60 bp InDel
SWlB 和 GATA29 通过 60 bp InDel 对染色质可及性和耐冷性的影响

To further elucidate the effects of SWIBs on chromatin accessibility associated with the 60 bp InDel fragment of WRKY34 promoter, we constructed SlSWIBb overexpressing lines driven by the CaMV 35S promoter (SlSWIBb-OE) and slswibab double mutants mediated by CRISPR/Cas9 in the background of LA4024 or LA3942. In an independent ATAC-qPCR assay, the region around the 60 bp InDel fragment of the WRKY34 promoter exhibited an increase in chromatin accessibility following cold treatment in LA3942 background (Fig. 6a). In contrast, the chromatin accessibility for this region did not change in response to cold stress in LA4024 background. Furthermore, in the slswibab double mutants of LA3942 background, the chromatin accessibility of this region was also lost in response to cold stress (Fig. 6a). Interestingly, overexpression of SlSWIBb in LA3942 background significantly increased chromatin accessibility of this region, and this region was more accessible under cold stress in LA3942 SlSWIBb-OE lines (Fig. 6a). RT-qPCR analysis revealed that WRKY34 did not respond to cold stress in the background of LA4024, consistent with the results of chromatin accessibility. Meanwhile, WRKY34 expression in LA3942 seedlings was significantly decreased after cold treatment, while WRKY34 expression in slswibab double mutants of LA3942 background remained unchanged regardless of cold treatment (Fig. 6b). Overexpression of SlSWIBb in LA3942 background did not affect the expression of WRKY34 under normal conditions. However, the expression of WRKY34 in SlSWIBb-OE lines of LA3942 background was significantly suppressed and lower than that in WT LA3942 seedlings after cold stress (Fig. 6b). Consistent with the expression of WRKY34, knockout of SlSWIBa and SlSWIBb or overexpression of SlSWIBb in LA4024 background had no effect on the cold tolerance of LA4024, as indicated by similar REL, Fv/Fm and survival rate (Fig. 6c–e and Supplementary Fig. 5c). However, slswibab double mutants of LA3942 exhibited compromised cold tolerance compared to WT, with higher REL, lower Fv/Fm and lower survival rate. In contrast, overexpression of SlSWIBb enhanced the cold tolerance of LA3942 resulting in lower REL, higher Fv/Fm and higher survival rate (Fig. 6c–e and Supplementary Fig. 5c).
为了进一步阐明 SWIBs 对与 WRKY34 启动子的 60 bp InDel 片段相关的染色质可及性的影响，我们在 LA4024 或 LA3942 的背景下构建了由 CaMV 35S 启动子 （SlSWIBb-OE） 和 CRISPR/Cas9 介导的 slswibab 双突变体驱动的 SlSWIBb 过表达系。在独立的 ATAC-qPCR 检测中，在 LA3942 背景中冷处理后，WRKY34 启动子的 60 bp InDel 片段周围的区域表现出染色质可及性增加（图 D）。相比之下，该区域的染色质可及性在 LA4024 背景中没有响应冷应激而改变。此外，在 LA3942 背景的 slswibab 双突变体中，该区域的染色质可及性也因冷应激而丢失（图 D）。有趣的是，在 LA3942 背景中过表达 SlSWIBb 显着增加了该区域的染色质可及性，并且在 LA3942 SlSWIBb-OE 系中，该区域在低温应激下更容易接近（图 D）。RT-qPCR 分析显示，WRKY34 在 LA4024 的背景下对冷应激没有反应，这与染色质可及性的结果一致。同时，冷处理后 LA3942 幼苗中 WRKY34 的表达显著降低，而 LA3942 背景的 slswibab 双突变体中 WRKY34 的表达无论冷处理如何都保持不变（图 D）。6b）. 在 LA3942 背景中过表达 SlSWIBb 在正常条件下不影响 WRKY34 的表达。 然而，在冷应激后，WRKY34 在 LA3942 背景的 SlSWIBb-OE 品系中的表达受到显著抑制，低于 WT LA3942 幼苗（图 D）。与 WRKY34 的表达一致，在 LA4024 背景中敲除 SlSWIBa 和 SlSWIBb 或过表达 SlSWIBb 对 LA4024 的耐冷性没有影响，如相似的 REL、Fv/Fm 和存活率所示（图 D）。6c–e 和补充图然而，与 WT 相比，LA3942 的 slswibab 双突变体表现出较低的耐冷性，具有更高的 REL 、更低的 Fv/Fm 和更低的存活率。相比之下，SlSWIBb 的过表达增强了 LA3942 的耐冷性，导致更低的 REL、更高的 Fv/Fm 和更高的存活率（图 D）。6c–e 和补充图5c）。

a ATAC-qPCR results from testing chromatin accessibility in the 60 bp InDel region of the WRKY34 promoter under cold stress in slswibab double mutants and SlSWIBb overexpressing (SlSWIBb-OE) seedlings of LA4024 or LA3942 background. Data are presented as means ± SD of three biological replicates. b Expression levels of WRKY34 in slswibab double mutants and SlSWIBb-OE seedlings of LA4024 or LA3942 background under cold stress. Total RNA was isolated from leaf samples after 6 h of cold stress. Data are presented as means ± SD of three biological replicates. The relative expression levels of WRKY34 in WT of LA4024 and LA3942 under normal conditions were set to “1”. c Phenotypes of slswibab double mutants and SlSWIBb-OE seedlings in LA4024 or LA3942 background at 7 d after cold treatment. Eight plants of each genotype and treatment were tested with similar results. Bar: 10 cm. Relative electrolyte leakage (REL, d) and maximum photochemical efficiency of photosystem II (Fv/Fm, e) in slswibab double mutants and SlSWIBb-OE seedlings of LA4024 or LA3942 background at 7 d after cold treatment. The false color code depicted at the bottom of the images ranges from 0 (black) to 1 (purple). Bar: 2 cm. Data are presented as means of four biological replicates ± SD (d) or means of eight leaflets from independent plants (e). f CRISPR/Cas9-induced deletions in the 60 bp InDel region of the WRKY34 promoter of LA3942. Blue-marked area represents PAM, green-marked area indicates SgRNA, yellow-marked area represents the 60 bp InDel, and red-marked area represents the SWIB-mu4/GATA-box. g ATAC-qPCR results from testing chromatin accessibility in the 60 bp InDel region of the WRKY34 promoter in response to cold stress in different types of 60 bp InDel deletion mutants. Data are presented as means ± SD of three biological replicates. All experiments were repeated twice with similar results. Different letters above bars indicate significant differences (P < 0.05) determined using one-way ANOVA with Duncan’s multiple range test. Source data are provided as a Source Data file.
a ATAC-qPCR 结果来自在 slswibab 双突变体和 LA4024 或 LA3942 背景的 SlSWIBb 过表达 （SlSWIBb-OE） 幼苗的低温胁迫下测试 WRKY34 启动子 60 bp InDel 区域的染色质可及性。数据以 3 个生物学重复± SD 平均值表示。b 低温胁迫下 WRKY34 在 slswibab 双突变体和 LA4024 或 LA3942 的 SlSWIBb-OE 幼苗中的表达水平。冷应激 6 小时后，从叶片样品中分离总 RNA。数据以 3 个生物学重复± SD 平均值表示。在正常条件下，LA4024 和 LA3942 在 WT 中 WRKY34 的相对表达水平设置为 “1”。c 冷处理后 7 d LA4024 或 LA3942 背景下 slswibab 双突变体和 SlSWIBb-OE 幼苗的表型。对每种基因型和处理的 8 株植物进行了测试，结果相似。杆：10 厘米。冷处理后 7 d lswibab 双突变体和 LA4024 或 LA3942 背景的 SlSWIBb-OE 幼苗的相对电解质泄漏 （REL， d） 和光系统 II （Fv/Fm， e） 的最大光化学效率。图像底部描绘的假色代码范围从 0（黑色）到 1（紫色）。杆：2 厘米。数据以 SD （d） ± 4 个生物学重复的平均值或来自独立植物的 8 个小叶 （e） 的平均值表示。f CRISPR/Cas9 诱导的 LA3942 WRKY34 启动子 60 bp InDel 区缺失。 蓝色标记区域代表 PAM，绿色标记区域表示 SgRNA，黄色标记区域代表 60 bp InDel，红色标记区域代表 SWIB-mu4/GATA-box。g ATAC-qPCR 结果来自测试 WRKY34 启动子的 60 bp InDel 区域的染色质可及性，以响应不同类型的 60 bp InDel 缺失突变体的冷应激。数据以 3 个生物学重复± SD 平均值表示。所有实验重复两次，结果相似。条形上方的不同字母表示使用单因素方差分析和 Duncan 多范围检验确定的显著差异 （P < 0.05）。源数据作为 源数据 文件提供。

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We also constructed SlGATA29 overexpressing lines driven by the CaMV 35 S promoter (SlGATA29-OE) and slgata29 mutants mediated by CRISPR/Cas9 in the background of LA4024 or LA3942. As shown in Supplementary Fig. 15a, the expression of WRKY34 showed no response to cold stress in LA4024 background. In contrast, the expression of WRKY34 in LA3942 background was significantly suppressed under cold stress, while cold-suppressed WRKY34 expression was compromised when SlGATA29 was knocked out in LA3942 background (Supplementary Fig. 15a). Conversely, overexpression of SlGATA29 further suppressed WRKY34 expression in LA3942 background under cold stress (Supplementary Fig. 15a). Consistent with the expression of WRKY34, SlGATA29 knockout or overexpression in LA4024 background had no effect on the cold tolerance of LA4024, as indicated by similar REL, Fv/Fm and survival rate (Supplementary Figs. 5d and 15b–d). However, slgata29 mutants of LA3942 exhibited compromised cold tolerance compared to WT, as indicated by higher REL, lower Fv/Fm and lower survival rate. In contrast, overexpression of SlGATA29 enhanced cold tolerance of LA3942, resulting in lower REL, higher Fv/Fm and higher survival rate (Supplementary Figs. 5d and 15b–d).
我们还构建了由 CaMV 35S 启动子 （SlGATA29-OE） 驱动的 SlGATA29 过表达系和在 LA4024 或 LA3942 背景下由 CRISPR/Cas9 介导的 slgata29 突变体。如补充图 1 所示。15a，WRKY34 的表达在 LA4024 背景下对冷应激无反应。相比之下，在低温胁迫下，LA3942 背景中 WRKY34 的表达受到显著抑制，而当 SlGATA29 在 LA3942 背景下敲除时，低温抑制的 WRKY34 表达受到影响（补充图 D）。相反，在低温胁迫下，SlGATA29 的过表达进一步抑制了 WRKY34 在 LA3942 背景中的表达（补充图 D）。与 WRKY34 的表达一致，SlGATA29 在 LA4024 背景中的敲除或过表达对 LA4024 的耐冷性没有影响，如相似的 REL、Fv/Fm 和存活率所示（补充图5d 和 15b-d）。然而，与 WT 相比，LA3942 的 slgata29 突变体表现出较低的耐冷性，如较高的 REL 、较低的 Fv/Fm 和较低的存活率所示。相比之下，SlGATA29 的过表达增强了 LA3942 的耐寒性，导致更低的 REL、更高的 Fv/Fm 和更高的存活率（补充图5d 和 15b-d）。

To explore the critical role of the 60 bp InDel in altering chromatin accessibility, we used the CRISPR/Cas9 to delete the 60 bp in the ShWRKY34 promoter of LA3942. We obtained two lines (named Cri-60bp-1 and Cri-60bp-2) with different deletions within the 60 bp InDel region (Fig. 6f). Specifically, Cri-60bp-1 exhibits a deletion of only 6 bp at the 3’ end of the 60 bp InDel, whereas Cri-60bp-2 has a more extensive deletion of 35 bp, which disrupts the crucial SWIB-mu4/GATA-box (Fig. 6f). As expected, ATAC-qPCR assays showed that compared with WT, the chromatin accessibility of the 60 bp InDel region was significantly weakened in Cri-60bp-2 under cold stress, while the chromatin accessibility of this region in Cri-60bp-1 was almost unchanged (Fig. 6g). Moreover, the repression effect of WRKY34 expression under cold stress was abolished in Cri-60bp-2, but not in Cri-60bp-1 (Supplementary Fig. 16a). Consistently, Cri-60bp-2 plants were more sensitive to cold stress than WT, exhibiting a significant increase in REL and a decrease in Fv/Fm under cold stress (Supplementary Fig. 16b–d). These observations confirm that the 60 bp InDel, regulated by SWIB and GATA29, is a major causal variation underlying the differential expression and chromatin accessibility of WRKY34 under cold stress.
为了探索 60 bp InDel 在改变染色质可及性中的关键作用，我们使用 CRISPR/Cas9 删除了 LA3942 的 ShWRKY34 启动子中的 60 bp。我们获得了两个细胞系（命名为 Cri-60bp-1 和 Cri-60bp-2），在 60 bp InDel 区域内具有不同的缺失（图 D）。具体来说，Cri-60bp-1 在 60 bp InDel 的 3' 端仅缺失 6 bp，而 Cri-60bp-2 在 60 bp InDel 的 3' 末端缺失更广泛，为 35 bp，这破坏了关键的 SWIB-mu4/GATA 盒（图 D）。6f）。正如预期的那样，ATAC-qPCR 检测显示，与 WT 相比，在低温胁迫下，Cri-60bp-2 中 60 bp InDel 区域的染色质可及性显著减弱，而 Cri-60bp-1 中该区域的染色质可及性几乎没有变化（图 D）。6g）。此外，WRKY34 在低温胁迫下表达的抑制作用在 Cri-60bp-2 中被消除，但在 Cri-60bp-1 中未被消除（补充图 D）。一致地，Cri-60bp-2 植物比 WT 对冷胁迫更敏感，在冷胁迫下表现出 REL 的显著增加和 Fv/Fm 的降低（补充图 D）。16b-d）。这些观察结果证实，受 SWIB 和 GATA29 调节的 60 bp InDel 是 WRKY34 在低温胁迫下差异表达和染色质可及性的主要因果变异。

WRKY34 disrupts the CBF-COR cold response pathway at both transcript and protein levels
WRKY34 在转录本和蛋白质水平上破坏 CBF-COR 冷反应通路

WRKY34 overexpression or mutation can influence the expression of CBFs and CORs in LA4024 after cold stress (Supplementary Fig. 6e). To further analyze whether WRKY34 affects the CBF-COR cold response pathway, we detected the protein interaction between WRKY34 and CBFs or CORs. Interestingly, SlWRKY34 can interact with SlCBF1 but not SlCBF2/3 or the other three SlCORs with its C terminal domain (Fig. 7a). Moreover, the C terminal domain of ShWRKY34 also interacted with ShCBF1 or SlCBF1, but not with ShCBF2/3, ShCORs, SlCBF2/3 and SlCORs (Supplementary Fig. 17). Using GST-pull down assays, we demonstrated that SlWRKY34-GST pulled down SlCBF1-His, while the negative control GST failed to do so (Fig. 7b). BiFC results showed that the full-length SlWRKY34 and SlCBF1 proteins interacted in the nucleus (Fig. 7c). Co-IP results revealed that SlWRKY34-HA associated with SlCBF1-GFP, but not with free GFP (Fig. 7d). These results confirm the interaction between SlWRKY34 and SlCBF1.
WRKY34 过表达或突变可影响冷应激后 LA4024 中 CBF 和 COR 的表达（补充图 D）。为了进一步分析 WRKY34 是否影响 CBF-COR 冷反应通路，我们检测了 WRKY34 与 CBFs 或 CORs 之间的蛋白质相互作用。有趣的是，SlWRKY34 可以与 SlCBF1 相互作用，但不能与 SlCBF2/3 或其他三个具有 C 末端结构域的 SlCOR 相互作用（图 D）。此外，ShWRKY34 的 C 末端结构域也与 ShCBF1 或 SlCBF1 相互作用，但不与 ShCBF2/3、ShCORs、SlCBF2/3 和 SlCORs 相互作用（补充图 D）。使用 GST 下拉测定法，我们证明 SlWRKY34-GST 下拉了 SlCBF1-His，而阴性对照 GST 未能做到这一点（图 D）。BiFC 结果表明，全长 SlWRKY34 和 SlCBF1 蛋白在细胞核中相互作用（图 7）。Co-IP 结果显示 SlWRKY34-HA 与 SlCBF1-GFP 相关，但不与游离 GFP 相关（图 D）。这些结果证实了 SlWRKY34 和 SlCBF1 之间的相互作用。

a Yeast two-hybrid assay (Y2H) testing the interactions of SlWRKY34 with SlCBF1 in yeast. SlW34-C, C terminal of SlWRKY34 protein. b Pull-down assays show that GST-tagged SlWRKY34 physically interacts with His-tagged SlCBF1. The green triangle indicates the target band of SlCBF1-His pulled down by SlW34-GST. The combination of GST and SlCBF1-His was used as a negative control. c Bimolecular fluorescence complementation assay (BiFC) showing the interactions between SlWRKY34 and SlCBF1 in tobacco. Bar: 50 μm. d Co-IP showing the interactions between SlWRKY34 and SlCBF1. The green triangle indicates the SlW34-HA target band immunoprecipitated with SlCBF1-GFP. The combination of GFP and SlW34-HA was used as a negative control. e EMSA show that SlWRKY34 interferes with the binding of SlCBF1 to DRE elements on downstream SlCBF1 and SlCOR47 promoters. Unlabeled wild type probes were used as cold probes. mu, mutated probes in which the DRE elements (5ʹ-G/ACCGAC-3ʹ) were replaced with 5ʹ-AAAAAA-3ʹ. f Transient dual-luciferase (dual-LUC) assay in tobacco leaves. Empty vector (EV) was included as control. Data are presented as means ± SD of six biological replicates. g Phenotypes of slwrky34 mutant in LA4024 background (slw34/LA4024) and its SlCBF1-silenced seedlings at 7 d after cold treatment. Eight plants of each genotype and treatment were tested with similar results. Bar: 10 cm. Relative electrolyte leakage (REL, h) and maximum photochemical efficiency of photosystem II (Fv/Fm, i) in slwrky34 mutant of LA4024 background (slw34/LA4024) and its SlCBF1-silenced seedlings at 7 d after cold treatment. The false color code depicted at the bottom of the images ranges from 0 (black) to 1 (purple). Bar: 2 cm. Data are presented as means of four biological replicates ± SD (h) or means of eight leaflets from independent plants (i). Experiments in (a–e) were repeated twice, and in (f–i), three times, all yielding similar results. Different letters above bars indicate significant differences (P < 0.05) determined using one-way ANOVA with Duncan’s multiple range test. TRV, non-silenced seedlings; TRV-SlCBF1, SlCBF1-silenced seedlings. Source data are provided as a Source Data file.
酵母双杂交测定 （Y2H） 测试 SlWRKY34 与 SlCBF1 在酵母中的相互作用。SlW34-C，SlWRKY34 蛋白的 C 末端。b Pull-down 测定显示 GST 标记的 SlWRKY34 与 His 标记的 SlCBF1 发生物理相互作用。绿色三角形表示 SlW34-GST 拉低的 SlCBF1-His 的目标条带。GST 和 SlCBF1-His 的组合用作阴性对照。c 双分子荧光互补试验 （BiFC） 显示烟草中 SlWRKY34 和 SlCBF1 之间的相互作用。棒：50 μm。d Co-IP 显示 SlWRKY34 和 SlCBF1 之间的相互作用。绿色三角形表示用 SlCBF1-GFP 免疫沉淀的 SlW34-HA 靶条带。GFP 和 SlW34-HA 的组合用作阴性对照。e EMSA 显示 SlWRKY34 干扰 SlCBF1 与下游 SlCBF1 和 SlCOR47 启动子上的 DRE 元件的结合。未标记的野生型探针用作冷探针。mu 突变探针，其中 DRE 元件 （5ʹ-G/ACCGAC-3ʹ） 被 5ʹ-AAAAAA-3ʹ 取代。f 烟叶中的瞬时双荧光素酶 （dual-LUC） 测定。空向量 （EV） 作为对照。数据以 6 次生物学重复的 SD ±平均值表示。g 冷处理后 7 d LA4024 背景 （slw34/LA4024） 及其 SlCBF1 沉默幼苗中 slwrky34 突变体的表型。对每种基因型和处理的 8 株植物进行了测试，结果相似。杆：10 厘米。冷处理后 7 d LA4024 背景 slwrky34 突变体 （slw34/LA4024） 及其 SlCBF1 沉默幼苗中光系统 II （Fv/Fm， i） 的相对电解质泄漏 （REL， h） 和光系统 II （Fv/Fm， i） 的最大光化学效率。 图像底部描绘的假色代码范围从 0（黑色）到 1（紫色）。杆：2 厘米。数据以 SD （h） ± 4 个生物学重复的平均值或来自独立植物的 8 个小叶 （i） 的平均值表示。（a-e） 中的实验重复两次，（f-i） 中的实验重复 3 次，都产生了相似的结果。条形上方的不同字母表示使用单因素方差分析和 Duncan 多范围检验确定的显著差异 （P < 0.05）。TRV，非沉默幼苗;TRV-SlCBF1，SlCBF1 沉默幼苗。源数据作为 源数据 文件提供。

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Many previous studies have shown that the CBF-COR pathway is central to plant cold tolerance³⁶. We hypothesized that SlWRKY34 interferes with the transcriptional function of SlCBF1 under cold stress by interacting with SlCBF1, thus weakening the cold tolerance of cultivated tomato. To test this hypothesis, we conducted EMSA and dual-LUC assays. As shown in Supplementary Fig. 18a, several CBF binding elements, known as Dehydration-Responsive Elements (DREs), were identified in the promoters of SlCBF1 and SlCOR47. SlCBF1 directly bound to DRE elements in SlCBF1 and SlCOR47 promoters in vitro; however, SlCBF1-bound probe signals decreased progressively with increasing concentrations of SlWRKY34 purified proteins (Fig. 7e). Additionally, SlCBF1 could transcriptionally activate the expression of itself and SlCOR47, while SlWRKY34 co-transfected with SlCBF1 significantly impaired SlCBF1-induced SlCBF1 and SlCOR47 expression (Fig. 7f). Furthermore, silencing SlCBF1 in slwrky34 mutants significantly reduced cold tolerance with higher REL and lower Fv/Fm under cold stress, compared with slwrky34 mutants (Fig. 7g–i).
许多先前的研究表明，CBF-COR 途径是植物耐寒性的核心³⁶。我们假设 SlWRKY34 通过与 SlCBF1 相互作用来干扰 SlCBF1 在低温胁迫下的转录功能，从而削弱了栽培番茄的耐寒性。为了检验这一假设，我们进行了 EMSA 和双 LUC 分析。如补充图 1 所示。18a，在 SlCBF1 和 SlCOR47 的启动子中鉴定出几种 CBF 结合元件，称为脱水反应元件 （DRE）。SlCBF1 在体外直接与 SlCBF1 和 SlCOR47 启动子中的 DRE 元件结合;然而，随着 SlWRKY34 纯化蛋白浓度的增加，SlCBF1 结合的探针信号逐渐减弱（图 D）。此外，SlCBF1 可以转录激活自身和 SlCOR47 的表达，而 SlWRKY34 与 SlCBF1 共转染显著损害 SlCBF1 诱导的 SlCBF1 和 SlCOR47 表达（图 1）。此外，与 slwrky34 突变体相比，在 slwrky34 突变体中沉默 SlCBF1 显著降低了冷耐受性，在冷应激下具有较高的 REL 和较低的 Fv/Fm （图 .7g-i）。

Moreover, we identified many W-box elements in SlCBFs and SlCOR47 promoters (Supplementary Fig. 18a). Next, we conducted Y1H and ChIP-qPCR assays, respectively. Y1H results showed that SlWRKY34 could directly bind to the promoters of SlCBFs and SlCOR47 in yeast cells (Supplementary Fig. 18b). ChIP-qPCR analysis showed that SlWRKY34 could directly bind to the promoters of SlCBFs and SlCOR47 in vivo under cold stress (Supplementary Fig. 18c). Furthermore, dual-LUC results indicated that SlWRKY34 could transcriptionally repress the expression of SlCBFs and SlCOR47 (Supplementary Fig. 18d). Thus, WRKY34 also directly suppresses gene transcription in the CBF-COR pathway.
此外，我们在 SlCBFs 和 SlCOR47 启动子中鉴定了许多 W-box 元件（补充图 D）。接下来，我们分别进行了 Y1H 和 ChIP-qPCR 检测。Y1H 结果表明，SlWRKY34 可以直接与酵母细胞中 SlCBFs 和 SlCOR47 的启动子结合（补充图 D）。ChIP-qPCR 分析表明，在低温胁迫下，SlWRKY34 可以在体内直接与 SlCBFs 和 SlCOR47 的启动子结合（补充图 D）。此外，双 LUC 结果表明，SlWRKY34 可以转录抑制 SlCBFs 和 SlCOR47 的表达（补充图 D）。因此，WRKY34 也直接抑制 CBF-COR 通路中的基因转录。

The tomato likely originated in the Andes mountains of South America and adjacent tropical regions³⁷. The diverse range of climatic and ecological conditions present across these areas has been instrumental in driving the diversification of tomatoes and their botanical relatives. Phylogenetic analyses have classified wild tomatoes into several groups: “Lycopersicon group” (S. pimpinellifolium, S. cheesmaniae, and S. galapagense), “Arcanum group” (S. arcanum, S. chmielewskii, and S. neorickii), “Eriopersicon group” (S. habrochaites, S. huaylasense, S. corneliomulleri, S. peruvianum, and S. chilense), “Neolycopersicon group” (S. pennellii); and two outgroups: Section Juglandifolia (S. juglandifolium and S. ochranthum) and Section Lycopersicoides (S. lycopersicoides and S. sitiens)³⁸. Each group adapts to specific altitudes and average temperatures, reflecting the influence of environmental factors on their evolutionary paths³⁸. In this study, we found a significant correlation between a 60 bp InDel in the WRKY34 promoter and WRKY34 expression under cold stress (Fig. 3). Specifically, the WRKY34 promoter in cultivated tomatoes, cherry tomatoes and “Lycopersicon group” of wild tomatoes exhibited this 60 bp deletion, while over 90% of other wild tomato groups contained the 60 bp insertion (Fig. 3d, e). Heatmap analysis revealed that the expression of WRKY34 variant with the 60 bp deletion showed no significant change post cold stress, whereas WRKY34 variant with the 60 bp insertion exhibited a marked decrease under cold stress (Supplementary Fig. 10). Notably, two wild species, S. cheesmaniae and S. galapagense, did not contain the 60 bp insertion (Fig. 3e and Supplementary Fig. 10), possibly due to their warm growing environment in low-altitude areas^39,40. The S. habrochaites introgression line LA3942, which contains a single introgression fragment where ShWRKY34 replaces SlWRKY34, shows better cold tolerance than its recurrent parent S. lycopersicum LA4024 (Fig. 2). Thus, the full 60 bp InDel can be introduced into cultivated and cherry tomatoes through backcrossing and other breeding technologies to improve their cold tolerance.
西红柿可能起源于南美洲的安第斯山脉和邻近的热带地区³⁷。这些地区存在的各种气候和生态条件有助于推动西红柿及其植物亲缘种的多样化。系统发育分析将野生西红柿分为几组：“石松组”（S. pimpinellifolium、S. cheesmaniae 和 S. galapagense）、“Arcanum 组”（S. arcanum、S. chmielewskii 和 S. neorickii）、“Eriopersicon 组”（S. habrochaites、S. huaylasense、S. corneliomulleri、S. peruvianum 和 S. chilense）、“新石松组”（S. pennellii);和两个外群：Juglandifolia Section （S. juglandifolium 和 S. ochranthum） 和 Lycopersicoides Section （S. lycopersicoides 和 S. sitiens）³⁸。每个群体都适应特定的海拔高度和平均温度，反映了环境因素对其进化路径的影响³⁸。在这项研究中，我们发现 WRKY34 启动子中 60 bp 的 InDel 与冷应激下的 WRKY34 表达之间存在显著相关性（图 D）。具体来说，栽培番茄、樱桃番茄和野生番茄的“石松组”中的 WRKY34 启动子表现出 60 bp 的缺失，而超过 90% 的其他野生番茄组包含 60 bp 的插入（图 D）。3d， e） 的 热图分析显示，具有 60 bp 缺失的 WRKY34 变体的表达在冷应激后没有显着变化，而具有 60 bp 插入的 WRKY34 变体在冷应激下表现出显着降低（补充图 D）。值得注意的是，两个野生物种 S. cheesmaniae 和 S. galapagense 不包含 60 bp 的插入（图 D）。3e 和补充图10），可能是由于它们在低海拔地区温暖的生长环境^39,40。沙门氏菌渗入系 LA3942 包含一个渗入片段，其中 ShWRKY34 取代了 SlWRKY34，显示出比其递归亲本石松石罗氏菌 LA4024 更好的耐寒性（图 1）。因此，完整的 60 bp InDel 可以通过回交和其他育种技术引入栽培番茄和樱桃番茄中，以提高它们的耐寒性。

Unlike changes in coding sequences, variations in cis-regulatory regions can alter gene expression in response to environmental cues and developmental processes without changing the protein they encode⁶. During evolution and domestication, certain variations in cis-regulatory regions can confer advantageous traits, such as enhanced yields, stress tolerance, or nutrient content. For instance, research on cotton (Gossypium hirsutum) has identified changes in cis-regulatory regions that have significantly altered gene expression during domestication, contributing to the development of desirable fiber traits⁴¹. One specific study identified a 26 bp InDel in the 5’ UTR of ZmGLK36, which modulated its expression and thus the plant’s resistance to maize rough dwarf virus (RBSDV), highlighting a key genetic adaptation for crop improvement in the face of disease challenges⁴². Previously, we found that a key W-box single nucleotide polymorphism (SNP) affects the self-transcriptional regulation and protein accumulation of WRKY33 under cold stress in cultivated tomato, thus contributing to the cold sensitivity of cultivated tomatoes compared with wild tomatoes⁷. Here, we found that the WRKY34 promoter in cold-tolerant wild tomato species contains a 60 bp insertion, which directly causes its chromatin to open and recruits the transcriptional suppressor GATA29 under cold stress, thereby diminishing WRKY34 expression and enhancing cold tolerance. Conversely, the absence of this 60 bp segment in the WRKY34 promoter of cultivated tomatoes leads to a reduced response to cold stress, contributing to the cold sensitivity observed in these domesticated tomato varieties. Both of our studies discovered SNP or InDel variations within the promoter regions of WRKY family transcription factors, leading to changes in gene expression levels and thereby affecting cold tolerance in tomatoes. Specifically, the mutation in WRKY33 resulted in the loss of a promoter cis-element during tomato evolution, while the mutation in WRKY34 involved the deletion of a fragment within the promoter. Although the functions and mechanisms of WRKY33 and WRKY34 in response to cold stress are completely different, variations in their promoters both result in decreased cold tolerance in cultivated tomato. Therefore, natural variation in the multiple genes related to cold tolerance may have occurred during the evolution of cultivated tomatoes and were preserved during domestication, resulting in an overall phenotype of cold sensitivity in cultivated tomatoes. Future research should explore how technologies such as gene editing can restore these natural variations and improve cold tolerance in cultivated tomatoes without altering other traits.
与编码序列的变化不同，顺式调节区的变化可以响应环境线索和发育过程改变基因表达，而不会改变它们编码的蛋白质⁶。在进化和驯化过程中，顺式调控区域的某些变化可以赋予有利的性状，例如提高产量、抗逆性或营养成分含量。例如，对棉花 （Gossypium hirsutum） 的研究已经确定了顺式调控区域的变化，这些变化在驯化过程中显着改变了基因表达，有助于理想纤维性状的发育⁴¹。一项具体研究在 ZmGLK36 的 5' UTR 中确定了 26 bp 的 InDel，它调节了其表达，从而调节了植物对玉米粗矮病毒 （RBSDV） 的抗性，突出了面对疾病挑战时作物改良的关键遗传适应⁴²。以前，我们发现关键的 W-box 单核苷酸多态性 （SNP） 影响培育番茄在低温胁迫下 WRKY33 的自我转录调控和蛋白质积累，从而导致培育番茄与野生番茄相比对寒冷的敏感性⁷。在这里，我们发现耐寒野生番茄物种中的 WRKY34 启动子包含一个 60 bp 的插入片段，这直接导致其染色质打开并在低温胁迫下募集转录抑制因子 GATA29，从而减少 WRKY34 表达并增强耐寒性。 相反，栽培番茄的 WRKY34 启动子中缺少这个 60 bp 片段，导致对冷应激的反应降低，从而导致在这些驯化番茄品种中观察到的冷敏感性。我们的两项研究都发现了 WRKY 家族转录因子启动子区域内的 SNP 或 InDel 变异，导致基因表达水平发生变化，从而影响西红柿的耐寒性。具体来说，WRKY33 中的突变导致番茄进化过程中启动子顺式元件的丢失，而 WRKY34 中的突变涉及启动子内片段的缺失。尽管 WRKY33 和 WRKY34 响应寒冷胁迫的功能和机制完全不同，但它们启动子的变化都会导致栽培番茄的耐寒性降低。因此，与耐寒性相关的多个基因的自然变异可能发生在栽培番茄的进化过程中，并在驯化过程中被保留下来，导致栽培番茄的整体表型为冷敏感。未来的研究应探索基因编辑等技术如何在不改变其他性状的情况下恢复这些自然变异并提高栽培西红柿的耐寒性。

The role of SWIB/MDM2 domain-containing proteins in chromatin remodeling is increasingly recognized^43,44. Typically found in SWI/SNF chromatin remodeling complexes, the SWIB/MDM2 domain is known to regulate gene transcriptional activity by altering nucleosome positioning⁴³. This regulation can either be specific, affecting certain genes, or broadly influencing chromatin states across the genome. However, our study reveals a more direct and precise mechanism of SWIB/MDM2 domain proteins in gene regulation. We observed that under normal conditions, SWIBs were inactive, not opening chromatin or recruiting transcription factors, resulting in the low expression of WRKY34. Nevertheless, under cold stress, SWIBs not only opened chromatin and recruited the transcription factor GATA29, but also directly bound to specific sites in the WRKY34 promoter, thereby precisely inducing chromatin opening in specific regions and recruiting transcription factor GATA29 to bind to the GATA-box within the 60 bp region, further suppressing the expression of the WRKY34 gene (Figs. 4–6). Even more excitingly, through protein structure prediction and experimental validation, we have demonstrated that three evolutionarily conserved amino acids in SWIBs are involved in DNA binding, with the key DNA binding site being TGATAA. We hypothesize that proteins of this family can recognize the TGATAA motif and then form a helical tripod structure to wrap around DNA, thereby further exerting their function. Therefore, the discovery about SWIBs extends beyond the traditional understanding of their role in altering nucleosome positions through chromatin remodeling and recruiting transcription factors. It reveals that these proteins can also directly bind to specific DNA sites through three evolutionarily conserved amino acids. This direct DNA binding capacity allows SWIB/MDM2 domain proteins to be key players in activating genes in chromatin-closed regions, enhancing the precision of specific gene expression control. Moreover, it demonstrates the ability of SWIB/MDM2 domain proteins to respond to specific environmental signals and regulate gene expression by acting directly on specific DNA elements, further emphasizing their versatility and importance in gene regulation. Beyond direct impacts on gene transcription, SWIB/MDM2 domain proteins are closely related to histone modifications and epigenetic regulation⁴⁵. It has been reported that SWIB domain proteins might directly recognize and bind to specific histones or their modified forms, thereby altering nucleosome stability or regulating interactions with other chromatin-associated proteins. For example, under normal conditions, SWP73A repressed NLR (NOD-like receptor) gene RPS2 through H3K9me2 modification, while this repression reduced or eliminated following pathogen infection, facilitating gene transcription and the activation of plant innate immunity⁴⁶. However, the association of the SWIB/MDM2 domain proteins in our study with histone modifications warrants further investigation.
包含 SWIB/MDM2 结构域的蛋白在染色质重塑中的作用越来越得到认可^43,44。SWIB/MDM2 结构域通常存在于 SWI/SNF 染色质重塑复合物中，已知通过改变核小体定位来调节基因转录活性⁴³。这种调节可以是特异性的，影响某些基因，也可以广泛影响整个基因组的染色质状态。然而，我们的研究揭示了 SWIB/MDM2 结构域蛋白在基因调控中的更直接和精确的机制。我们观察到在正常情况下，SWIBs 无活性，不打开染色质或募集转录因子，导致 WRKY34 低表达。然而，在低温胁迫下，SWIBs 不仅打开染色质并募集转录因子 GATA29，还直接结合到 WRKY34 启动子中的特定位点，从而精确诱导特定区域的染色质开放，并募集转录因子 GATA29 与 60 bp 区域内的 GATA-box 结合，进一步抑制 WRKY34 基因的表达（图4-6）。更令人兴奋的是，通过蛋白质结构预测和实验验证，我们已经证明 SWIBs 中的三个进化上保守的氨基酸参与了 DNA 结合，其中关键的 DNA 结合位点是 TGATAA。我们假设该家族的蛋白质可以识别 TGATAA 基序，然后形成螺旋三脚架结构包裹 DNA，从而进一步发挥其功能。因此，关于 SWIB 的发现超越了对其通过染色质重塑和募集转录因子改变核小体位置的作用的传统理解。 它揭示了这些蛋白质也可以通过三个进化上保守的氨基酸直接与特定的 DNA 位点结合。这种直接的 DNA 结合能力使 SWIB/MDM2 结构域蛋白成为激活染色质封闭区域基因的关键参与者，从而提高特异性基因表达控制的精度。此外，它证明了 SWIB/MDM2 结构域蛋白通过直接作用于特定 DNA 元件来响应特定环境信号和调节基因表达的能力，进一步强调了它们在基因调控中的多功能性和重要性。除了对基因转录的直接影响外，SWIB/MDM2 结构域蛋白还与组蛋白修饰和表观遗传调控密切相关⁴⁵。据报道，SWIB 结构域蛋白可能直接识别并结合特定组蛋白或其修饰形式，从而改变核小体稳定性或调节与其他染色质相关蛋白的相互作用。例如，在正常情况下，SWP73A 通过 H3K9me2 修饰抑制 NLR（NOD 样受体）基因 RPS2，而这种抑制在病原体感染后减少或消除，促进基因转录和植物先天免疫的激活⁴⁶。然而，我们研究中 SWIB/MDM2 结构域蛋白与组蛋白修饰的关联值得进一步研究。

Research on WRKY34 in Arabidopsis indicates that it is specifically expressed in pollen, negatively regulates the cold tolerance of mature pollen, and may be involved in the CBF signal cascade in mature pollen⁴⁷. In our study, WRKY34 also negatively impacts cold tolerance in tomato seedlings, primarily by interfering with the classic CBF-COR cold response pathway at both transcription and protein levels (Fig. 7 and Supplementary Fig. 18). WRKY34 can directly bind to W-box elements in the promoters of CBFs and CORs, transcriptionally repressing their expression. Additionally, WRKY34 interacts with CBF1, disrupting its transcriptional activation of itself and downstream CORs. Notably, while knocking out WRKY34 enhanced cold tolerance, the wrky34 mutants exhibited developmental defects, such as smaller fruits and fewer seeds per fruit than WT (Supplementary Fig. 7). Moreover, the expression and protein accumulation of WRKY34 were the highest in roots, followed by flowers and buds, with lower expression and protein accumulation in leaves and fruits (Supplementary Fig. 8). This emphasizes WRKY34’s necessity under normal conditions and suggests that complete functional loss isn’t a desirable improvement approach for cold tolerance. Gene functions are multifaceted. Although knocking out or overexpressing genes can achieve desired traits, it may also disrupt other characteristics, like growth and development. Precisely regulating gene transcription through specific promoter control is vital in crop breeding, as this approach effectively balances the enhancement of desired traits with the overall health and growth of the plant. For example, a recent study utilized a gene-editing strategy targeting the SlPIF4 binding motif in the SlCOMT2 promoter, effectively enhancing melatonin levels in tomato fruit during the ripening stage without impacting other developmental phases. In addition, this targeted approach achieved higher melatonin content and no growth defects compared to pif4 knockout mutants, demonstrating the efficacy of precise genetic modulation in crop development⁴⁸. Here, we identified a 60 bp insertion in the WRKY34 promoter that diminished its expression under cold stress in Solanum species, thus boosting cold tolerance. Through multiple generations of backcrossing, we have also developed the ShWRKY34 introgression tomato line LA3942, which exhibits cold tolerance without impacting other traits. Additionally, considering that variations in cis-regulatory regions typically exert subtler phenotypic impacts and circumvent the adverse effects of coding region mutations, we advocate employing gene-editing techniques to incorporate this 60 bp sequence into the WRKY34 promoter of Solanum plants lacking this sequence.
拟南芥中 WRKY34 的研究表明，它在花粉中特异性表达，负向调节成熟花粉的耐寒性，并可能参与成熟花粉⁴⁷ 中的 CBF 信号级联反应。在我们的研究中，WRKY34 还对番茄幼苗的耐寒性产生负面影响，主要是通过在转录和蛋白质水平上干扰经典的 CBF-COR 冷反应途径（图 D）。7 和补充图WRKY34 可以直接与 CBFs 和 CORs 启动子中的 W-box 元件结合，转录抑制它们的表达。此外，WRKY34 与 CBF1 相互作用，破坏其自身和下游 COR 的转录激活。值得注意的是，在敲除 WRKY34 增强的耐寒性时，WRKY34 突变体表现出发育缺陷，例如比 WT 更小的果实和更少的果实种子（补充图 D）。7）. WRKY34 在根中的表达和蛋白质积累最高，其次是花和芽，在叶和果实中的表达和蛋白质积累较低 （补充图 D）。这强调了 WRKY34 在正常条件下的必要性，并表明完全功能丧失并不是耐寒性的理想改进方法。基因功能是多方面的。尽管敲除或过表达基因可以获得所需的性状，但它也可能破坏其他特性，如生长和发育。通过特异性启动子控制精确调节基因转录在作物育种中至关重要，因为这种方法有效地平衡了所需性状的增强与植物的整体健康和生长。 例如，最近的一项研究利用了一种靶向 SlCOMT2 启动子中 SlPIF4 结合基序的基因编辑策略，在成熟阶段有效提高了番茄果实中的褪黑激素水平，而不会影响其他发育阶段。此外，与 pif4 敲除突变体相比，这种靶向方法实现了更高的褪黑激素含量和无生长缺陷，证明了精确遗传调控在作物发育中的功效⁴⁸。在这里，我们在 WRKY34 启动子中鉴定了一个 60 bp 的插入，该插入在冷应激下降低了其在茄属物种中的表达，从而提高了耐寒性。通过多代回交，我们还开发了 ShWRKY34 渗入番茄系 LA3942，它在不影响其他性状的情况下表现出耐寒性。此外，考虑到顺式调控区的变化通常会产生更微妙的表型影响并规避编码区突变的不利影响，我们提倡采用基因编辑技术将这个 60 bp 序列掺入缺乏该序列的茄属植物的 WRKY34 启动子中。

Accessibility is generally positively correlated with expression, but examples of increased chromatin accessibility and decreased gene expression have been reported. For example, by investigating chromatin modifications and accessibility, a study suggests that although type A ARF exhibits an open chromatin configuration, it is regulated by a network of transcriptional repressors⁴⁹. Therefore, the relationship between chromatin accessibility and gene expression is complex and influenced by multiple factors. Here, our results demonstrate one of these mechanisms and we thus propose a working model of WRKY34-mediated cold tolerance in wild and cultivated tomatoes (Fig. 8). Under cold stress, the presence of a 60 bp insertion in the WRKY34 promoter of wild tomato S. habrochaites leads to the binding of chromatin remodeling factor SWIBs, thereby opening chromatin in the nearby region and recruiting transcriptional repressor GATA29 to bind to the GATA-box within the 60 bp, resulting in repression of WRKY34 expression. WRKY34 interferes with CBF1-induced expression of itself and CORs by interacting with CBF1. Furthermore, WRKY34 directly binds to the promoters of downstream CBFs and CORs and represses their expression under cold stress. However, the deletion of the 60 bp DNA fragment in the WRKY34 promoter of cultivated tomatoes results in its inability to bind SWIBs under cold stress, preventing chromatin opening and recruitment of GATA29, and thus failing to suppress WRKY34 expression and contributing to the cold sensitivity of these tomatoes. Three additional points deserve to be mentioned. Firstly, ATAC-Seq data indicates that wild tomato LA1777 exhibits more chromatin opening under cold stress than cultivated tomato AC, suggesting potential functional differences in chromatin remodeling factors other than SWIBs between wild and cultivated tomatoes, which may impact cold tolerance. Secondly, in addition to the regulatory differences caused by non-coding regions, the differences in coding regions between wild and cultivated tomatoes and their potential impact on resistance traits warrant further investigation.
可及性通常与表达呈正相关，但已报道了染色质可及性增加和基因表达降低的例子。例如，通过调查染色质修饰和可及性，一项研究表明，尽管 A 型 ARF 表现出开放的染色质构型，但它受转录抑制因子网络的调节⁴⁹。因此，染色质可及性与基因表达之间的关系很复杂，并受多种因素影响。在这里，我们的结果证明了其中一种机制，因此我们提出了一个 WRKY34 介导的野生和栽培西红柿耐寒工作模型（图 D）。8）. 在低温胁迫下，野生番茄 S. habrochaites 的 WRKY34 启动子中存在 60 bp 插入导致染色质重塑因子 SWIBs 的结合，从而在附近区域打开染色质并募集转录抑制因子 GATA29 与 60 bp 内的 GATA 盒结合，导致 WRKY34 表达的抑制。WRKY34 通过与 CBF1 相互作用干扰 CBF1 诱导的自身和 CORs 表达。此外，WRKY34 直接与下游 CBFs 和 COR 的启动子结合，并在低温应激下抑制它们的表达。然而，培养番茄 WRKY34 启动子中 60 bp DNA 片段的缺失导致其在低温胁迫下无法结合 SWIBs，阻止染色质打开和 GATA29 募集，从而无法抑制 WRKY34 表达，并导致这些番茄对低温敏感。还有三点值得一提。 首先，ATAC-Seq 数据表明，野生番茄 LA1777 在低温胁迫下比培养番茄 AC 表现出更多的染色质开放，这表明野生番茄和培育番茄之间除 SWIB 以外的染色质重塑因子存在潜在的功能差异，这可能会影响耐寒性。其次，除了非编码区引起的调控差异外，野生番茄和栽培番茄之间编码区的差异及其对抗性状的潜在影响值得进一步研究。

The presence of the 60 bp InDel in the WRKY34 promoter of wild tomato S. habrochaites leads to the binding of chromatin remodeling factor SWIBs, thereby opening chromatin in the nearby region, and recruiting more transcriptional repressor GATA29 to bind to the GATA-box within the 60 bp, resulting in repression of WRKY34 expression. In addition, WRKY34 interferes with the transcriptional regulation of CBF1 to itself and CORs by interacting with CBF1. On the other hand, WRKY34 directly binds downstream CBFs and CORs promoters and represses their expression under cold stress. However, the deletion of the 60 bp DNA fragment in the WRKY34 promoter of cultivated tomatoes results in its inability to bind SWIBs under cold stress, preventing chromatin opening and recruitment of GATA29, and thus failing to suppress WRKY34 expression and contributing to the cold sensitivity of these tomatoes. Figure 8 Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
野生番茄 S. habrochaites 的 WRKY34 启动子中存在 60 bp InDel 导致染色质重塑因子 SWIB 的结合，从而在附近区域打开染色质，并募集更多的转录抑制因子 GATA29 与 60 bp 内的 GATA 盒结合，导致 WRKY34 表达的抑制。此外，WRKY34 通过与 CBF1 相互作用来干扰 CBF1 对自身和 COR 的转录调控。另一方面，WRKY34 直接结合下游 CBFs 和 CORs 启动子，并在低温应激下抑制它们的表达。然而，培养番茄 WRKY34 启动子中 60 bp DNA 片段的缺失导致其在低温胁迫下无法结合 SWIBs，阻止染色质打开和 GATA29 募集，从而无法抑制 WRKY34 表达，并导致这些番茄对低温敏感。图 8 使用在 Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International 许可下发布的 BioRender.com 创建。

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Plant materials and growth conditions
植物材料和生长条件

Wild tomato (S. habrochaites accession LA1777) and cultivated tomato (S. lycopersicum cv. Ailsa Craig, AC) were used for RNA-Seq and ATAC-Seq. The S. habrochaites introgression line LA3942, containing a single introgression fragment with ShWRKY34 replacing SlWRKY34, along with its recurrent parent S. lycopersicum LA4024 and donor parent LA1777, was used for virus-induced gene silencing (VIGS) of WRKY34 genes. LA4024 and LA3942 were selected for genetic transformation.
野生番茄 （S. habrochaites accession LA1777） 和栽培番茄 （S. lycopersicum cv. Ailsa Craig， AC） 用于 RNA-Seq 和 ATAC-Seq。沙棘血吸虫渗入系 LA3942 包含一个渗入片段，其中 ShWRKY34 取代了 SlWRKY34，以及其递归亲本石松菌 LA4024 和供体亲本 LA1777，用于病毒诱导的 WRKY34 基因基因沉默 （VIGS）。选择 LA4024 和 LA3942 进行遗传转化。

A total of 376 tomato accessions were collected from various sources, including Tomato Genetics Resource Center (TGRC), United State Department of Agriculture (USDA), University of Florida, and European Union Solanaceae Project (EU-SOL). These accessions include 63 wild tomato accessions (3 S. cheesmaniae, 2 S. galapagense, 8 S. arcanum, 3 S. chmielewskii, 9 S. neorickii, 3 S. huaylasense, 4 S. corneliomulleri, 4 S. peruvianum, 12 S. chilense, 10 S. habrochaites, 4 S. pennellii and 1 S. sitien), 58 S. pimpinellifolium, 74 S. lycopersicum var. cerasiforme, and 181 S. lycopersicum accessions (Supplementary Data 6).
共从各种来源收集了 376 份番茄种质，包括番茄遗传学资源中心 （TGRC）、美国农业部 （USDA）、佛罗里达大学和欧盟茄科项目 （EU-SOL）。这些种质包括 63 个野生番茄种质（3 个 S. cheesmaniae、2 个 S. galapagense、8 个 S. arcanum、3 个 S. chmielewskii、9 个 S. neorickii、3 个 S. huaylasense、4 个 S. corneliomulleri、4 个 S. peruvianum、12 个 S. chilense、10 个 S. habrochaites、4 个 S. pennellii 和 1 个 S. sitien）、58 个 S. pimpinellifolium、74 个 S. lycopersicum var. cerasiforme 和 181 个 S. lycopersicum种质（补充数据 6）。

Seeds were germinated on moistened filter paper at 28 °C in the dark and subsequently sown in 72-cell plastic flats filled with a mixture of peat and vermiculite (3:1, v:v). Upon reaching the two-leaf stage, seedlings were transplanted into plastic pots (10 cm × 10 cm in height × diameter, one seedling per pot) or 32-cell plastic flats containing the same medium. The plants were cultivated in a growth room under a 12 h photoperiod, with temperature of 25/20°C (day/night), and a photosynthetic photon flux density (PPFD) of 600 μmol m⁻² s⁻¹. The relative humidity was maintained at 70%, and plants were irrigated with 1/2 strength Hoagland’s nutrient solution every 3 d.
种子在 28 °C 的黑暗中在湿润的滤纸上发芽，随后播种在装满泥炭和蛭石混合物的 72 细胞塑料平板中（3：1，v：v）。达到两叶阶段后，将幼苗移植到塑料盆（高 × 高 10 厘米×直径，每盆一棵幼苗）或含有相同培养基的 32 细胞塑料平板中。植物在 12 小时光周期下在生长室中培养，温度为 25/20°C（昼/夜），光合光子通量密度 （PPFD） 为 600 μmol ^{m-2 s-1}。相对湿度保持在 70%，每 3 d 用 1/2 强度的 Hoagland's 营养液灌溉植株。

Cold stress treatment and cold tolerance evaluation
冷应激处理和耐寒性评价

For cold stress treatment, tomato seedlings at the five-leaf stage or tobacco plants expressing reporter vectors were transferred to a cold artificial growth chamber set at 4 °C, maintaining the same conditions as in the growth room. Each biological repeat contained eight seedlings from each tomato genotype, with three biological repeats per treatment. After 7 d of cold treatment, tomato seedlings were photographed. Then, relative electrolyte leakage (REL) was measured based on electrical conductivity and the maximum photochemical efficiency of photosystem II (Fv/Fm) was measured using an Imaging-PAM Chlorophyll Fluorometer equipped with a computer-operated PAM-control unit (IMAG-MAXI; Heinz Walz, Effeltrich, Germany), as previously described⁵⁰. Survival rate assays were conducted on 20-day-old seedlings (at the three-leaf stage) grown in the growth room, which were subjected to 4 °C treatment for the specified duration before being returned to normal conditions (25 °C) for 1 week of recovery. During this process, the survival rate (percentage of green plants recovered after cold treatment) was calculated. The survival rates of the seedlings were calculated with three independent replicates for each genotype.
对于冷胁迫处理，将五叶期的番茄幼苗或表达报告基因载体的烟草植株转移到设定为 4 °C 的低温人工生长室中，保持与生长室相同的条件。每个生物重复包含来自每个番茄基因型的 8 个幼苗，每个处理有 3 个生物重复。冷处理 7 d 后，拍摄番茄幼苗。然后，基于电导率测量相对电解质泄漏 （REL），并使用配备计算机操作的 PAM 控制单元 （IMAG-MAXI;Heinz Walz， Effeltrich， Germany），如前所述⁵⁰.对生长室中生长的 20 日龄幼苗 （三叶期） 进行存活率测定，这些幼苗在 4 °C 下处理规定的时间，然后恢复到正常条件 （25 °C） 进行 1 周的恢复。在此过程中，计算存活率（冷处理后恢复的绿色植物的百分比）。幼苗的存活率是通过每种基因型的 3 个独立重复来计算的。

RNA-Seq libraries preparation and data analysis
RNA-Seq 文库制备和数据分析

RNA-Seq was performed as previously described⁷. Briefly, tomato leaves of AC and LA1777 were collected under normal conditions or after 6 h of cold stress, respectively, and used for RNA extraction. RNA-Seq library preparation and paired-end sequencing were performed on an Illumina Novaseq^TM 6000 sequence platform by LC Sciences (Hangzhou, China). Approximately 4 Gb of high-quality paired-end reads were generated from each library. Clean data (clean reads) were obtained by removing reads containing adapters, poly-N sequences and low-quality reads from raw data using Trimmomatic version 0.36. These clean reads were then aligned to the tomato genome (https://solgenomics.net, SL4.0) using the Hisat2 mapping tool. Genes with FPKM’s P < 0.05 and an absolute log₂-fold change ≥ 1 were considered as differentially expressed genes (DEGs).
如前所述进行 RNA-Seq⁷。简而言之，分别在正常条件下或冷应激 6 h 后收集 AC 和 LA1777 的番茄叶片，用于 RNA 提取。RNA-Seq 文库制备和双端测序由 LC Sciences（中国杭州）在 Illumina Novaseq^TM 6000 序列平台上进行。每个文库生成了大约 4 Gb 的高质量双端读长。通过使用 Trimmomatic 0.36 版从原始数据中删除包含接头、poly-N 序列和低质量读数的读数来获得干净数据（干净读数）。然后使用 Hisat2 定位工具将这些干净的读数与番茄基因组 （https://solgenomics.net，SL4.0） 比对。FPKM 的 P < 0.05 且绝对对数变化≥_{1 的 2} 倍变化的基因被认为是差异表达基因 （DEGs）。

Nuclei extraction and purification
细胞核提取和纯化

Samples were prepared using sucrose sedimentation as previously reported⁵¹ but with slight modifications. Briefly, young leaves of AC and LA1777 were collected under normal conditions or after 6 h of cold stress, respectively, and ground to fine powder in liquid nitrogen. For each sample, 0.2 g of frozen tissue powder was homogenized in prechilled 1 ml lysis buffer (15 mM Tris-HCl pH7.5, 20 mM NaCl, 80 mM KCl, 0.5 mM spermine, 5 mM 2-ME, 0.2% TritonX-100), and the nuclear fraction was purified as described⁵². The nuclei pellet was resuspended in 1 ml cold lysis buffer. For ATAC-Seq and ATAC-qPCR, a nuclei aliquot (25 µl) was stained with DAPI (10 µl of 1 µg ml^-1) and counted using a haemocytometer. Approximately 50,000 nuclei were used for each ATAC-Seq or ATAC-qPCR reaction.
使用蔗糖沉降制备样品，如前所述⁵¹，但略有修改。简单来说，在正常条件下或冷应激 6 h 后分别收集 AC 和 LA1777 的幼叶，并在液氮中研磨成细粉。对于每个样品，将 0.2 g 冷冻组织粉末在预冷的 1 ml 裂解缓冲液（15 mM Tris-HCl pH7.5、20 mM NaCl、80 mM KCl、0.5 mM 精胺、5 mM 2-ME、0.2% TritonX-100）中匀浆，并按所述⁵² 纯化核级分。将细胞核沉淀重悬于 1 ml 冷裂解缓冲液中。对于 ATAC-Seq 和 ATAC-qPCR，用 DAPI（10 μl 1 μg ^{ml -1} ）对细胞核等分试样 （25 μl） 进行染色，并使用血细胞计数器计数。每个 ATAC-Seq 或 ATAC-qPCR 反应使用大约 50,000 个细胞核。

ATAC-Seq libraries preparation and data analysis
ATAC-Seq 文库制备和数据分析

ATAC-Seq was carried out as previously described⁵³ with some minor modifications. Briefly, nuclei were extracted and purified from samples, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37 °C for 30 min. Equimolar Adapter1 and Adatper2 were added after transposition, and PCR was then performed to amplify the library. After the PCR reaction, libraries were purified with AMPure beads (Beckman, A63881) and library quality was assessed with Qubit (Thermo Fisher, Q32854). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform at Novogene (Beijing, China) and 150 bp paired-end reads were generated.
ATAC-Seq 如前所述⁵³ 进行，并进行了一些细微的修改。简而言之，从样品中提取和纯化细胞核，并将细胞核沉淀重悬于 Tn5 转座酶反应混合物中。转座反应在 37 °C 下孵育 30 分钟。转座后加入 Equimolar Adapter1 和 Adatper2，然后进行 PCR 以扩增文库。PCR 反应后，用 AMPure 珠子 （Beckman， A63881） 纯化文库，用 Qubit （Thermo Fisher， Q32854） 评估文库质量。根据制造商的说明，使用 TruSeq PE Cluster Kit v3-cBot-HS （Illumina） 在 cBot Cluster Generation System 上对索引编码样本进行聚类。生成簇后，在 Novogene（中国北京）的 Illumina 平台上对文库制备进行测序，并生成 150 bp 的双端读长。

Raw data was processed using fastp (version 0.20.0) to obtain clean reads, excluding adapters, poly-N, and low-quality sequences, while calculating Q20, Q30, and GC content. The reference genome and annotation were downloaded (https://solgenomics.net, SL4.0), and its index was built with BWA (version 0.7.12) for alignment of clean reads. Reads from mitochondria and chloroplast DNA, improperly paired, and PCR duplicates were excluded. Peak calling was done with MACS2 (version 2.1.0). By default, peaks with q-value threshold of 0.05 were carried out for all datasets. Peaks of different groups were merged using ‘bedtools merge’. We calculated the mean RPM of each group in the merge peak. Only peaks with an absolute log₂-fold change of RPM ≥ 1 and P < 0.05 were considered as differential peaks. Genes associated with different peaks were identified using ChIPseeker. ChIPseeker was also used for gene and genomic region annotation⁵⁴. GO enrichment analysis and KEGG pathway analysis were performed⁵⁵. Differential peaks were identified with fold change of RPM more than 2. Genes associated with different peaks were identified using ChIPseeker. Peaks were visualized using the Integrative Genomics Viewer (version 2.12.2).
使用 fastp（0.20.0 版）处理原始数据，以获得干净的读数，不包括接头、poly-N 和低质量序列，同时计算 Q20、Q30 和 GC 含量。下载参考基因组和注释 （https://solgenomics.net， SL4.0），并使用 BWA （版本 0.7.12） 构建其索引以对齐干净读数。排除了线粒体和叶绿体 DNA 的读数，配对不正确，并且 PCR 重复。峰值调用是使用 MACS2（版本 2.1.0）完成的。默认情况下，对所有数据集执行 q 值阈值为 0.05 的峰值。使用 'bedtools merge' 合并不同组的峰。我们计算了合并峰中每组的平均 RPM。只有 RPM ≥ 1 和 P < 0.05 绝对对数变化₂ 倍的峰才被视为差异峰。使用 ChIPseeker 鉴定与不同峰相关的基因。ChIPseeker 也用于基因和基因组区域注释⁵⁴。进行 GO 富集分析和 KEGG 通路分析⁵⁵。鉴定出 RPM 倍数变化大于 2 的差异峰。使用 ChIPseeker 鉴定与不同峰相关的基因。使用 Integrative Genomics Viewer（2.12.2 版）对峰进行可视化。

ATAC-qPCR

ATAC-qPCR was performed using the SYBR Green PCR Master Mix Kit (Takara, Shiga, Japan) on a Light Cycler 480 II detection system (Roche, Basel, Switzerland). Primers used for this analysis are shown in Supplementary Table 1. The relative accessibility and standard errors were determined using the 2^−ΔΔCT method⁵⁶.
使用 SYBR Green PCR 预混液试剂盒（Takara，Shiga，Japan）在 Light Cycler 480 II 检测系统（Roche，Basel，瑞士）上进行 ATAC-qPCR。用于该分析的引物如补充表 1 所示。使用 2^−ΔΔCT 方法确定相对可及度和标准误差⁵⁶。

VIGS VIGS （水）

Complementary DNA (cDNA) fragments of target genes were amplified using gene-specific primers containing EcoRI and BamHI restriction sites (Supplementary Table 2). Purified PCR products were cloned into the TRV2 vector. The plasmids were then transformed into Agrobacterium tumefaciens strain GV3101. Fully expanded cotyledons of tomato seedlings were infiltrated with a mixture of A. tumefaciens strain carrying the helper vector TRV1 mixed at 1:1 with the strain carrying either TRV2 (empty vector control, TRV) or TRV2-target gene vectors⁵⁷. The infiltrated plants were maintained in the growth chambers, and the silencing efficiency of the targeted genes was determined by RT-qPCR (Supplementary Fig. 19).
使用含有 EcoRI 和 BamHI 限制性位点的基因特异性引物扩增靶基因的互补 DNA （cDNA） 片段（补充表 2）。将纯化的 PCR 产物克隆到 TRV2 载体中。然后将质粒转化到根癌农杆菌菌株 GV3101 中。用携带辅助载体 TRV1 的根癌曲霉菌株的混合物浸润番茄幼苗的完全膨胀子叶，该菌株以 1：1 的比例混合，该菌株携带 TRV2（空载体对照，TRV）或 TRV2 靶基因载体⁵⁷。将浸润的植物维持在生长室中，并通过 RT-qPCR 测定目标基因的沉默效率（补充图 D）。19）.

Constructs for genetic transformation
用于遗传转化的构建体

For overexpression constructs, the full-length coding sequences (CDS) of SlWRKY34, SlGATA29 and SlSWIBb were amplified from LA4024 cDNA and the CDS of ShWRKY34 was amplified from LA1777 cDNA using specific primers (Supplementary Data 8). For generating transgenic overexpressing lines, the SlWRKY34, ShWRKY34 and SlGATA29 CDS were inserted into a pFGC1008-3HA binary plasmid vector behind the CaMV 35S promoter. The SlSWIBb CDS was inserted into a pAC402-GFP binary plasmid vector behind the CaMV 35S promoter. All vectors were transformed into A. tumefaciens strain GV3101 for plant transformation. The resulting SlWRKY34 and ShWRKY34 overexpression plasmids were introduced into cultivated tomato LA4024, while both LA4024 and LA3942 were used as transgenic recipient materials to transform SlGATA29 and SlSWIBb overexpression plasmids. The transgenic overexpressing lines were further identified by RT-qPCR (Supplementary Fig. 20). The homozygous T2 transgenic lines were used in subsequent studies.
对于过表达构建体，使用 LA4024 cDNA 从 LA4024 cDNA 中扩增 SlWRKY34 、 SlGATA29 和 SlSWIBb 的全长编码序列 （CDS），并从 LA1777 cDNA 中扩增 ShWRKY34 的 CDS（补充数据 8）。为了产生转基因过表达系，将 SlWRKY34、ShWRKY34 和 SlGATA29 CDS 插入 CaMV 35S 启动子后面的 pFGC1008-3HA 二元质粒载体中。将 SlSWIBb CDS 插入 CaMV 35S 启动子后面的 pAC402-GFP 二元质粒载体中。将所有载体转化到根癌不动杆菌菌株 GV3101 中用于植物转化。将所得的 SlWRKY34 和 ShWRKY34 过表达质粒引入培养番茄 LA4024 中，同时 LA4024 和 LA3942 均用作转基因受体材料，转化 SlGATA29 和 SlSWIBb 过表达质粒。通过 RT-qPCR 进一步鉴定转基因过表达系（补充图 1）。20）. 纯合 T2 转基因系用于后续研究。

For the CRISPR/Cas9 constructs, single-guide (sgRNAs) or two-guide RNAs containing 20-bp targeting sequences were designed using the CRISPR-P web tool (http://cbi.hzau.edu.cn/crispr/) (Supplementary Data 8). The synthesized sequences were annealed and inserted into the BbsI site of the AtU6-sgRNA-AtUBQ-Cas9 vector as previously described⁵⁸. The resulting plasmids were digested by HindIII and KpnI and then inserted into the pCAMBIA1301 binary vector digested by the same restriction enzymes. All resulting plasmids were transformed into A. tumefaciens strain GV3101 and infected into tomato cotyledons. First-generation transgenic plants were genotyped with specific primers surrounding the target sites (Supplementary Table 3). The homozygous F2 mutant lines without Cas9 were selected and used for further study. As shown in Supplementary Fig. 21a, the slwrky34-4 mutants harbored a 1 bp insertion and slwrky34-5 a 2 bp deletion in the SlWRKY34 coding region, leading to early translation termination. The protein bands of WRKY34 are barely observable in wrky34 mutants (Supplementary Fig. 21b). The slswibab double mutants in LA4024 background harbored a 4 bp deletion in the SlSWIBa coding region and a 1 bp deletion in the SlSWIBb coding region, both leading to early translation termination (Supplementary Fig. 21c). The slswibab double mutants in LA3942 background harbored a 1 bp deletion in the SlSWIBa coding region and a 4 bp deletion in the SlSWIBb coding region, both leading to early translation termination (Supplementary Fig. 21c). The slgata29 mutants in LA4024 background harbored a 2 bp deletion in the SlGATA29 coding region and the slgata29 mutants in LA3942 background harbored an 8 bp deletion in the SlGATA29 coding region, both leading to early translation termination (Supplementary Fig. 21d).
对于 CRISPR/Cas9 构建体，使用 CRISPR-P 网络工具 （http://cbi.hzau.edu.cn/crispr/） 设计包含 20 bp 靶向序列的单向导 （sgRNA） 或双向导 RNA（补充数据 8）。如前所述，将合成的序列退火并插入 AtU6-sgRNA-AtUBQ-Cas9 载体的 BbsI 位点⁵⁸。所得质粒经 HindIII 和 KpnI 消化，然后插入到由相同限制性内切酶消化的 pCAMBIA1301 二元载体中。所有得到的质粒均转化根癌曲霉菌株 GV3101 并感染番茄子叶。第一代转基因植物用靶位点周围的特异性引物进行基因分型（补充表 3）。选择不含 Cas9 的纯合 F2 突变株系并用于进一步研究。如补充图 1 所示。21a，slwrky34-4 突变体在 SlWRKY34 编码区携带 1 bp 插入和 slwrky34-5 2 bp 缺失，导致早期翻译终止。WRKY34 的蛋白质条带在 wrky34 突变体中几乎无法观察到（补充图 D）。LA4024 背景中的 slswibab 双突变体在 SlSWIBa 编码区存在 4 bp 缺失，在 SlSWIBb 编码区具有 1 bp 缺失，均导致翻译提前终止（补充图 D）。21c）. LA3942 背景中的 slswibab 双突变体在 SlSWIBa 编码区含有 1 bp 缺失，在 SlSWIBb 编码区含有 4 bp 缺失，均导致翻译提前终止（补充图 D）。21c）。 LA4024 背景中的 slgata29 突变体在 SlGATA29 编码区携带 2 bp 缺失，LA3942 背景中的 slgata29 突变体在 SlGATA29 编码区携带 8 bp 缺失，均导致翻译提前终止（补充图 1）。21d）。

RNA isolation and RT-qPCR
RNA 分离和 RT-qPCR

Tomato leaves were collected under normal conditions or after indicated hours of cold stress. Other environmental conditions (e.g., humidity, lighting) of the growth chambers were kept consistent during sample collection. Total RNA was extracted from tomato leaves using an RNAprep Pure Plant Kit (Tiangen, DP419) following the manufacturer’s instructions. DNase I-treated extracted RNA (2 μg) was reverse-transcribed using a ReverTra Ace qPCR RT Kit (Vazyme, R223). For RT-qPCR, quantitative PCR was performed using the SYBR Green PCR Master Mix Kit (Vazyme, Q711) on a Light Cycler 480 II detection system (Roche, Basel, Switzerland). Tomato housekeeping genes Actin2 and Ubiquitin3 were used as internal references. Relative gene expression was calculated as previously described⁵⁶. Primers used for RT-qPCR are listed in Supplementary Data 9.
在正常条件下或在指定数小时的冷应激后收集番茄叶。在样品采集过程中，生长室的其他环境条件 （例如湿度、照明） 保持一致。按照制造商的说明，使用 RNAprep 纯植物试剂盒 （Tiangen， DP419） 从番茄叶中提取总 RNA。使用 ReverTra Ace qPCR RT 试剂盒 （Vazyme， R223） 逆转录 DNase I 处理的提取 RNA （2 μg）。对于 RT-qPCR，在 Light Cycler 480 II 检测系统（Roche，Basel，瑞士）上使用 SYBR Green PCR 预混液试剂盒 （Vazyme， Q711） 进行定量 PCR。番茄看家基因 Actin2 和 Ubiquitin3 用作内部参考。如前所述计算相对基因表达⁵⁶。用于 RT-qPCR 的引物列于补充数据 9 中。

DNA extraction, PCR and sequencing
DNA 提取、PCR 和测序

Genomic DNAs of different tomato varieties were extracted using the TIANamp Genomic DNA Kit (Tiangen, DP304) and stored at -80°C. The nucleotide sequences of the 60 bp InDel in WRKY34 promoters of different tomato varieties were amplified with the following general primers: F: 5’-TGATATGAAAACCATTCACAAGTTGA-3’; R: 5’- TAGGGTGGTGAAAATGAGGTACATA-3’. The amplified products were sequenced by Sanger sequencing using an ABI 3730xl instrument by Youkang Biotechnology (Hangzhou, China). The sequencing results are shown in Supplementary Data 6.
使用 TIANamp 基因组 DNA 试剂盒 （Tiangen， DP304） 提取不同番茄品种的基因组 DNA，并在 -80°C 下储存。 用以下通用引物扩增不同番茄品种 WRKY34 启动子中 60 bp InDel 的核苷酸序列：F：5'-TGATATGAAAACCATTCACAAGTTGA-3';R： 5'- TAGGGTGGTGAAAATGAGGTACATA-3'.使用 Youkang Biotechnology（中国杭州）的 ABI 3730xl 仪器通过 Sanger 测序对扩增产物进行测序。测序结果显示在补充数据 6 中。

Transient expression assays in tobacco leaves
烟叶中的瞬时表达测定

Transient expression assays in tobacco leaves were performed as previously described in ref. ⁷. For promoter activity assays, promoters of pSlW34, pShW34, pSlW34^+60bp, pShW34^mW-box, pShW34^mGATA-box and pSlW34^+30bp were inserted into pGreenII 0800-LUC vectors as reporter genes. Renilla luciferase (REN) gene driven by CaMV 35S promoter in pGreenII 0800-LUC was used as an internal control to quantify transformation efficiency. Then, the above constructs were transformed into A. tumefaciens strain GV3101 and infiltrated into three-week-old tobacco leaves. After inoculation for 36 h, tobacco plants were treated at 4 °C for 6 h and proteins were extracted using Dual Luciferase Reporter Assay Kit (Vazyme, DL101).
如参考文献 ⁷ 中所述，在烟叶中进行瞬时表达测定。对于启动子活性测定，将 pSlW34、pShW34、pSlW34^+60bp、pShW34^mW-box、pShW34^mGATA-box 和 pSlW34^+30bp 的启动子作为报告基因插入 pGreenII 0800-LUC 载体中。使用 pGreenII 0800-LUC 中 CaMV 35S 启动子驱动的海肾荧光素酶 （任） 基因作为内部对照来定量转化效率。然后，将上述构建体转化到根癌不动杆菌菌株 GV3101 中，并渗透到 3 周龄的烟叶中。接种 36 小时后，将烟草植物在 4 °C 下处理 6 小时，并使用双荧光素酶报告基因检测试剂盒 （Vazyme， DL101） 提取蛋白质。

For dual-luciferase (LUC) transcription activity assays, full-length CDSs of SlGATA29, ShGATA29, SlSWIBa/b, ShSWIB, SlCBF1 and SlWRKY34 were inserted into pGreenII 0029 62-SK vectors as effectors. The empty vector SK was used as a control. Promoters of pSlW34, pShW34, pSlW34^+60bp, pShW34^mW-box, pShW34^mGATA-box, pSlW34^+30bp, pSlCBF1 and pSlCOR47 were inserted into pGreenII 0800-LUC vectors as reporter genes. Then, all the constructs were transformed into A. tumefaciens strain GV3101. The tobacco leaves were transfected with different combinations of vectors (A. tumefaciens strain carrying the pGreenII 0800-LUC vector or pGreenII 0029 62-SK vector in a 1:10 ratio) for 36 h, then collected and lysed for the detection of dual luciferase activity (Vazyme, DL101) according to the manufacturer’s recommendations.
对于双荧光素酶 （LUC） 转录活性测定，将 SlGATA29、ShGATA29、SlSWIBa/b、ShSWIB、SlCBF1 和 SlWRKY34 的全长 CDS 作为效应子插入 pGreenII 0029 62-SK 载体中。空向量 SK 用作对照。将 pSlW34 、 pShW34 、 pSlW34^+60bp 、 pShW34^mW-box 、 pShW34^mGATA-box 、 pSlW34 ^{+ 30bp} 、 pSlCBF1 和 pSlCOR47 的启动子作为报告基因插入 pGreenII 0800-LUC 载体中。然后，将所有构建体转化到根癌曲霉菌株 GV3101 中。用不同的载体组合（携带 pGreenII 0800-LUC 载体或 pGreenII 0029 62-SK 载体的根癌曲霉菌株，比例为 1：10）转染烟叶 36 小时，然后收集并裂解以检测双荧光素酶活性 （Vazyme， DL101） 根据制造商的建议。

The activities of firefly LUC and renilla luciferase REN were measured using the Glomax 96 microplate luminometer (Promega, Fitchburg, USA). The measured levels were normalized by calculating the LUC/REN ratio. Primers used for plasmid construction are listed in Supplementary Data 8.
使用 Glomax 96 微孔板发光计（Promega，Fitchburg，USA）测量萤火虫 LUC 和海肾荧光素酶 任 的活性。通过计算 LUC/任 比率对测得的水平进行归一化。用于质粒构建的引物列在补充数据 8 中。

Y1H assay Y1H 测定

Y1H assays were performed as previously described⁵⁹. The 60 bp InDel was amplified and cloned into the pAbAi vector to generate pAbAi-60bp, while the 60 bp InDel containing mutant GATA-box or SWIB-mu4 was also amplified and cloned into the pAbAi vector to generate pAbAi-mut^GATA-box or pAbAi-mut^SWIB-mu4. The promoters of SlCBF1/2/3 and SlCOR47 were amplified and cloned into the pAbAi vector to generate pAbAi-SlCBF1, pAbAi-SlCBF2, pAbAi-SlCBF3 and pAbAi-SlCOR47. Full-length CDSs of SlGATA29, ShGATA29, SlSWIBa, SlSWIBb, ShSWIB and SlWRKY34 were amplified and cloned into the pGADT7 vector as prey plasmids. The mutant SlSWIBa/b CDSs (SlSWIBa^R6A, SlSWIBa^L46A, SlSWIBa^K88A, SlSWIBa^allmut, SlSWIBb^R6A, SlSWIBb^L44A, SlSWIBb^K86A, and SlSWIBb^allmut) were synthesized and cloned into the pGADT7 vector by Youkang Biotechnology (Hangzhou, China). The linearized pAbAi constructs were transformed into Y1HGold yeast strain as bait strains, and screened with different Aureobasidin A (AbA) concentrations to detect background AbAr expression of bait strains. Then, prey plasmids were transformed into bait strains, and the transformed yeast cells were selected on selective plates (SD-Leu) supplemented with 150 ng ml⁻¹ or 200 ng ml⁻¹ AbA. The empty pGADT7 was used as the negative control. Primers used for plasmid construction are listed in Supplementary Data 8.
如前所述进行 Y1H 测定⁵⁹。扩增 60 bp InDel 并克隆到 pAbAi 载体中以生成 pAbAi-60bp，同时含有突变体 GATA-box 或 SWIB-mu4 的 60 bp InDel 也被扩增并克隆到 pAbAi 载体中以生成 pAbAi-mut^GATA-box 或 pAbAi-mut^SWIB-mu4。扩增 SlCBF1/2/3 和 SlCOR47 的启动子并克隆到 pAbAi 载体中，生成 pAbAi-SlCBF1、pAbAi-SlCBF2、pAbAi-SlCBF3 和 pAbAi-SlCOR47。扩增 SlGATA29 、 ShGATA29 、 SlSWIBa 、 SlSWIBb 、 ShSWIB 和 SlWRKY34 的全长 CDS 并克隆到 pGADT7 载体中作为猎物质粒。由优康生物科技（中国杭州）合成突变体 SlSWIBa/b CDSs （SlSWIBa^R6A、SlSWIBa^L46A、SlSWIBa^K88A、SlSWIBa^allmut、SlSWIBb^R6A、SlSWIBb^L44A、SlSWIBb^K86A 和 SlSWIBb^allmut） 并克隆到 pGADT7 载体中。将线性化的 pAbAi 构建体转化到 Y1HG 酵母菌株中作为诱饵菌株，并用不同浓度的 Aureobasidin A （AbA） 筛选以检测诱饵菌株的背景 AbAr 表达。然后，将猎物质粒转化成诱饵菌株，并在补充有 150 ng ^ml-1 或 200 ng ^ml-1 AbA 的选择性板 （SD-Leu） 上选择转化的酵母细胞。空 pGADT7 用作阴性对照。用于质粒构建的引物列在补充数据 8 中。

Recombinant proteins and electrophoretic mobility shift assays (EMSA)
重组蛋白和电泳迁移率变化测定 （EMSA）

Full-length CDSs of SlGATA29, ShGATA29, SlSWIBa, SlSWIBb, ShSWIB, SlCBF1 and SlWRKY34 were PCR amplified and cloned into the pET-28a vector (Supplementary Data 8). All recombinant vectors were transformed into Escherichia coli strain BL21 (DE3) and expressed at 37°C until OD₆₀₀ reached 0.6, and then induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, SIGMA, 092M4001V) at 16 °C for 14 h. The recombinant His-fusion proteins were purified according to the instructions provided with the Novagen pET purification system. To carry out EMSAs, oligonucleotide probes (Supplementary Table 4) were biotin-labelled using the Biotin 3′-End DNA Labeling Kit (Thermo Fisher Scientific, 89818) according to the manufacturer’s protocol and annealed to double-stranded DNA. EMSAs were performed using the Light Shift Chemiluminescent EMSA kit (Thermo Fisher Scientific, 20148) according to the manufacturer’s instructions⁶⁰. Briefly, purified recombinant proteins were incubated with biotin-labelled probes at 28 °C for 30 min in 20 μl binding buffer (10× binding buffer, 50% glycerol, 25 ng μl⁻¹ poly-dI-dC, 1% NP-40). For competition assays, 10-, 20- or 100-fold non-labelled competitor DNA was added to the reaction. The reaction products were resolved on a 6% polyacrylamide gels in 0.5 × TBE at 100 V for 1-2 h on ice. Probe-protein complexes and free probes were transferred to a charged Hybond-N membrane and detected by western blotting with 1:5000 diluted anti-biotin antibodies (Abcam, ab53494).
对 SlGATA29、ShGATA29、SlSWIBa、SlSWIBb、ShSWIB、SlCBF1 和 SlWRKY34 的全长 CDS 进行 PCR 扩增并克隆到 pET-28a 载体中（补充数据 8）。将所有重组载体转化到大肠杆菌菌株 BL21 （DE3） 中，在 37°C 下表达至 OD₆₀₀ 达到 0.6，然后在 16 °C 下用 0.5 mM 异丙基 β-D-1-硫代吡喃半乳糖苷 （IPTG， SIGMA， 092M4001V） 诱导 14 h。根据 Novagen pET 纯化系统提供的说明纯化重组 His 融合蛋白。为了进行 EMSA，根据制造商的方案，使用生物素 3′-末端 DNA 标记试剂盒 （Thermo Fisher Scientific， 89818） 对寡核苷酸探针（补充表 4）进行生物素标记，并退火为双链 DNA。根据制造商的说明⁶⁰，使用 Light Shift 化学发光 EMSA 试剂盒（Thermo Fisher Scientific，20148）进行 EMSA。简而言之，将纯化的重组蛋白与生物素标记的探针在 28 °C 下在 20 μl 结合缓冲液（1×0 μl 结合缓冲液、50% 甘油、25 ng ^μl-1 poly-dI-dC、1% NP-40）中孵育 30 分钟。对于竞争检测，向反应中加入 10 倍、20 倍或 100 倍的未标记竞争者 DNA。反应产物在 6% 聚丙烯酰胺凝胶中，在 0.5 × TBE 中于 100 V 下在冰上分离 1-2 小时。将探针-蛋白质复合物和游离探针转移到带电的 Hybond-N 膜上，并使用 1：5000 稀释的抗生物素抗体（Abcam，ab53494）通过蛋白质印迹法进行检测。

Microscale thermophoresis (MST) assay
微量热泳 （MST） 测定

The mutant SlSWIBa/b CDSs (SlSWIBa^R6A, SlSWIBa^L46A, SlSWIBa^K88A, SlSWIBa^allmut, SlSWIBb^R6A, SlSWIBb^L44A, SlSWIBb^K86A, and SlSWIBb^allmut) were synthesized and cloned into the pET-28a vector by Youkang Biotechnology (Hangzhou, China). All recombinant His-proteins were induced and purified as described above. The Cy5-labeled double-stranded DNA was synthesized and diluted by MST buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 0.05% tween-20. 16 micro reaction tubes were set up, with tube 1 containing His-tag SlSWIBa/b proteins and tubes 2 to 16 filled with MST buffer. A dilution process was initiated by transferring a sample from tube 1 to tube 2. A serial dilution was obtained by repeating 15 times and remove 10 μl from tube number 16 after mixing. 10 μl of Cy5-labeled DNA were mixed with 10 μl of purified proteins and incubated at room temperature for 10 minutes, then loaded into silica capillaries (Polymicro Technologies, TSP010150). Binding reactions were measured using a Monolith NT.115 instrument (NanoTemper Technologies) at 25 °C, 40% MST power and 20% LED power. The Kd values were calculated using the mass action equation via the NanoTemper MO. Affinity Analysis software (NanoTemper Technologies).
突变体 SlSWIBa/b CDSs （SlSWIBa^R6A、SlSWIBa^L46A、SlSWIBa^K88A、SlSWIBa^allmut、SlSWIBb^R6A、SlSWIBb^L44A、SlSWIBb^K86A 和 SlSWIBb^allmut）由优康生物科技（中国杭州）合成并克隆到 pET-28a 载体中。如上所述，对所有重组 His 蛋白进行诱导和纯化。合成 Cy5 标记的双链 DNA，并用含有 50 mM Tris-HCl （pH 8.0）、150 mM NaCl 和 0.05% tween-20 的 MST 缓冲液稀释。设置 16 个微量反应管，其中 1 管含有 His 标签 SlSWIBa/b 蛋白，2 至 16 管填充有 MST 缓冲液。通过将样品从试管 1 转移到试管 2 来启动稀释过程。重复 15 次获得连续稀释，混合后从 16 号试管中取出 10 μl。将 10 μl Cy5 标记的 DNA 与 10 μl 纯化的蛋白质混合，在室温下孵育 10 分钟，然后加载到二氧化硅毛细管中（Polymicro Technologies，TSP010150）。使用 Monolith NT.115 仪器 （NanoTemper Technologies） 在 25 °C、40% MST 功率和 20% LED 功率下测量结合反应。通过 NanoTemper MO 使用质量作用方程计算 Kd 值。亲和力分析软件 （NanoTemper Technologies）。

ChIP-qPCR ChIP-qPCR 技术

ChIP experiments were performed using the EpiQuiK Plant ChIP kit (Epigentek, 50-109-6154) as described in the manufacturer’s protocol⁷. Approximately 1.5 g of leaf tissue was harvested from transgenic plants and WT plants under normal conditions or after 6 h of cold stress, respectively. The harvested tissues were crosslinked in 1× PBS buffer containing 1% formaldehyde for 15 min under vacuum. Fixation was stopped by adding 1× PBS buffer containing 0.125 M glycine under vacuum for 5 min. After washing three times with cold sterilized water, the tissues were homogenized, dried and ground into powder in liquid nitrogen, followed by isolation and sonication of chromatin. Sonicated chromatin fragments were immunoprecipitated with either anti-HA antibody or anti-GFP antibody, while a goat anti-mouse IgG antibody was served as the negative control. The enriched DNA was amplified by qPCR using specific primers (Supplementary Table 5). Relative enrichment was calculated by comparing the percentage of anti-HA- or anti-GFP-immunoprecipitated DNA to the percentage of IgG-immunoprecipitated DNA.
按照制造商的方案⁷ 中所述，使用 EpiQuiK 植物 ChIP 试剂盒 （Epigentek， 50-109-6154） 进行 ChIP 实验。在正常条件下或冷应激 6 小时后，分别从转基因植株和 WT 植株收获约 1.5 g 叶组织。将收获的组织在含有 1% 甲醛的 1× PBS 缓冲液中在真空下交联 15 分钟。通过在真空下加入 1× 含有 0.125 M 甘氨酸的 PBS 缓冲液 5 分钟终止固定。用冷消毒水洗涤 3 次后，将组织匀浆、干燥并在液氮中研磨成粉末，然后分离和超声处理染色质。超声处理的染色质片段用抗 HA 抗体或抗 GFP 抗体进行免疫沉淀，而山羊抗小鼠 IgG 抗体作为阴性对照。使用特异性引物通过 qPCR 扩增富集的 DNA（补充表 5）。通过将抗 HA 或抗 GFP 免疫沉淀 DNA 的百分比与 IgG 免疫沉淀 DNA 的百分比进行比较来计算相对富集。

Y2H assay Y2H 测定

The CDSs of SlSWIBa, SlSWIBb, ShSWIB and the C-terminal fragment of SlWRKY34 and ShWRKY34 were amplified and cloned into the pGBKT7 vector. The CDSs of SlGATA29, ShGATA29, SlCBF1/2/3, ShCBF1/2/3, SlCOR47, SlCOR15a, SlCOR27, ShCOR47, ShCOR15a and ShCOR27 were amplified and cloned into the pGADT7 vector. The resulting constructs were co-transformed in various combinations into Saccharomyces cerevisiae strain AH109 according to the manufacture’s instruction (Yeastmaker™ Yeast Transformation System 2). The transfected yeast cells were grown on SD-Leu-Trp plates at 28 °C for 3 d and then transferred to selective plates (SD-Leu/-Trp/-His/-Ade) at 28 °C for 4 d. Primers used for plasmid construction are listed in Supplementary Data 8.
扩增 SlSWIBa 、 SlSWIBb 、 ShSWIB 的 CDS 以及 SlWRKY34 和 ShWRKY34 的 C 端片段并克隆到 pGBKT7 载体中。扩增 SlGATA29 、 ShGATA29 、 SlCBF1/2/3 、 ShCBF1/2/3 、 SlCOR47 、 SlCOR15a 、 SlCOR27 、 ShCOR47 、 ShCOR15a 和 ShCOR27 的 CDS 并克隆到 pGADT7 载体中。根据制造商的说明（Yeastmaker™ 酵母转化系统 2），将所得构建体以各种组合共转化到酿酒酵母菌株 AH109 中。将转染的酵母细胞在 28 °C 的 SD-Leu-Trp 平板上生长 3 d，然后在 28 °C 下转移至选择性平板 （SD-Leu/-Trp/-His/-Ade） 上 4 d。用于质粒构建的引物列在补充数据 8 中。

Pull-down assay Pull-down 测定

Full-length CDSs of SlSWIBa, SlSWIBb, ShSWIB and SlCBF1 were amplified and cloned into the pET-28a vector. Full-length CDSs of SlGATA29, ShGATA29 and SlWRKY34 were amplified and cloned into the pGEX-4T-3 vector. The recombinant vectors were transformed into E. coli BL21 (DE3). The recombinant His-fusion proteins were purified according to the instructions provided with the Novagen pET purification system. To carry out pull-down assays, GST and GST-fusion proteins were extracted with extraction buffer and kept immobilized on Glutathione Sepharose 4B beads (Cytiva, 17075601)⁶¹. Glutathione beads containing the GST and GST-fusion proteins were incubated with equal amounts of different His-fusion proteins at 4 °C for 3 h and then were washed five times with PBS buffer (containing 0.1% tween-20). The proteins were detected by immunoblotting with anti-His antibody and anti-GST antibody. Primers used for plasmid construction are listed in Supplementary Data 8.
扩增 SlSWIBa 、 SlSWIBb 、 ShSWIB 和 SlCBF1 的全长 CDS 并克隆到 pET-28a 载体中。扩增 SlGATA29 、 ShGATA29 和 SlWRKY34 的全长 CDS 并克隆到 pGEX-4T-3 载体中。将重组载体转化到大肠杆菌 BL21 （DE3） 中。根据 Novagen pET 纯化系统提供的说明纯化重组 His 融合蛋白。为了进行沉降测定，用提取缓冲液提取 GST 和 GST 融合蛋白，并将其固定在谷胱甘肽琼脂糖凝胶 4B 珠 （Cytiva， 17075601）⁶¹ 上。将含有 GST 和 GST 融合蛋白的谷胱甘肽珠与等量的不同 His 融合蛋白在 4 °C 下孵育 3 小时，然后用 PBS 缓冲液（含有 0.1% 吐温-20）洗涤五次。通过用抗 His 抗体和抗 GST 抗体免疫印迹检测蛋白质。用于质粒构建的引物列在补充数据 8 中。

Bimolecular fluorescence complementation (BiFC)
双分子荧光互补 （BiFC）

For BiFC assay, full-length CDSs of SlSWIBa, SlSWIBb, ShSWIB and SlWRKY34 were amplified and cloned into the binary vector p2YN. Full-length CDSs of SlGATA29, ShGATA29 and SlCBF1 were amplified and cloned into the binary vector p2YC. They were then transformed into A. tumefaciens strain GV3101. For transient expression, different combinations of A. tumefaciens carrying different constructs at OD₆₀₀ = 0.8 were co-infiltrated into four-week-old Nicotiana benthamiana leaves. Nucleus-located H2B-mCherry was used as a nucleus marker. After 36 h of infiltration, the fluorescence signals of the infiltrated leaves were observed under a confocal laser scanning microscope (Zeiss LSM 780, Oberkochen, Germany) using preset settings of YFP (Ex: 488 nm, Em: 520-540 nm) and mCherry (Ex: 561 nm, Em: 610-630 nm). Primers used for plasmid construction are listed in Supplementary Data 8.
对于 BiFC 测定，将 SlSWIBa 、 SlSWIBb 、 ShSWIB 和 SlWRKY34 的全长 CDS 扩增并克隆到二元载体 p2YN 中。扩增 SlGATA29 、 ShGATA29 和 SlCBF1 的全长 CDS 并将其克隆到二元载体 p2YC 中。然后将它们转化到根癌不动杆菌菌株 GV3101 中。对于瞬时表达，在 OD₆₀₀ = 0.8 时携带不同构建体的根癌曲霉的不同组合被共渗透到 4 周龄的本氏烟草叶中。位于细胞核的 H2B-mCherry 用作细胞核标记物。浸润 36 小时后，在共聚焦激光扫描显微镜（Zeiss LSM 780，Oberkochen，Germany）下使用 YFP （Ex： 488 nm， Em： 520-540 nm） 和 mCherry （Ex： 561 nm， Em： 610-630 nm） 的预设设置观察浸润叶子的荧光信号。用于质粒构建的引物列在补充数据 8 中。

Co-immunoprecipitation (Co-IP)
免疫共沉淀 （Co-IP）

Full-length CDSs of SlSWIBa, SlSWIBb, ShSWIB and SlCBF1 were amplified and cloned into GFP tag vector, while full-length CDSs of SlGATA29, ShGATA29 and SlWRKY34 were amplified and cloned into HA tag vector. They were then transformed into A. tumefaciens strain GV3101. For transient expression, different combinations of A. tumefaciens carrying different constructs at OD₆₀₀ = 0.8 were co-infiltrated into four-week-old N. benthamiana leaves. After 36 h of infiltration, the proteins co-expressed in the infiltrated leaves were extracted using Co-IP buffer and analyzed by immunoblotting with anti-HA antibody and anti-GFP antibody⁶⁰. Extracts of equal total proteins were incubated with anti-GFP Magnetic Beads (Chromotek) for 3 h with gentle rotation at 4°C. Beads were washed five times with the Co-IP buffer. The immunoprecipitated proteins were analyzed by immunoblotting with anti-HA antibody. Primers used for plasmid construction are listed in Supplementary Data 8.
将 SlSWIBa 、 SlSWIBb 、 ShSWIB 和 SlCBF1 的全长 CDS 扩增并克隆到 GFP 标签载体中，同时将 SlGATA29 、 ShGATA29 和 SlWRKY34 的全长 CDS 扩增并克隆到 HA 标签载体中。然后将它们转化到根癌不动杆菌菌株 GV3101 中。对于瞬时表达，在 OD₆₀₀ = 0.8 时携带不同构建体的根癌农杆菌的不同组合被共渗透到 4 周龄的本氏牛笼草叶片中。浸润 36 小时后，使用 Co-IP 缓冲液提取浸润叶片中共表达的蛋白质，并使用抗 HA 抗体和抗 GFP 抗体⁶⁰ 进行免疫印迹分析。将等总蛋白的提取物与抗 GFP 磁珠 （Chromotek） 一起在 4°C 下轻轻旋转孵育 3 小时。 用 Co-IP 缓冲液洗涤磁珠 5 次。通过抗 HA 抗体免疫印迹分析免疫沉淀的蛋白质。用于质粒构建的引物列在补充数据 8 中。

Protein extraction and western blotting
蛋白质提取和蛋白质印迹

Protein extraction and western blotting were performed as described^60,62. Proteins were separated by SDS-PAGE using 10% (w:v) acrylamide gels and then transferred onto nitrocellulose membranes. WRKY34 protein was detected with anti-WRKY34 polyclonal antibody (No.230608037). Anti-WRKY34 polyclonal antibody was customized by the Laboratory Animal Center of Zhejiang University.
如所述进行蛋白质提取和蛋白质印迹^60,62。使用 10% （w：v） 丙烯酰胺凝胶通过 SDS-PAGE 分离蛋白质，然后转印到硝酸纤维素膜上。用抗 WRKY34 多克隆抗体 （No.230608037） 检测 WRKY34 蛋白。抗 WRKY34 多克隆抗体由浙江大学实验动物中心定制。

Statistical analysis 统计分析

Data were subjected to statistical analysis of variance using the SPSS package (SPSS 19.0). Data were represented as the mean ± SD. The difference between the two groups was assessed by two tailed Student’s t-tests. Statistically significant differences among multiple groups were evaluated by one-way ANOVA followed by a Duncan’s multiple range test. Details of each statistical test are indicated in the figure legends.
使用 SPSS 软件包 （SPSS 19.0） 对数据进行方差统计分析。数据表示为 SD ±平均值。通过两个有尾的学生 t 检验评估两组之间的差异。通过单因素方差分析后跟 Duncan 多范围检验评估多组之间的统计学显着差异。图例中显示了每个统计测试的详细信息。

Reporting summary 报告摘要

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
有关研究设计的更多信息，请参阅本文链接的 Nature Portfolio Reporting Summary。